Menu
US
asgct-1920x400.jpg.jpg
Event

Revvity at ESGCT 2024

Discover Revvity @ESGCT 2024

Join Revvity at the ESGCT event as we drive the future of cell and gene therapy. Our cutting-edge tools and services are tailored to advance your research with unparalleled precision and efficiency. At Revvity, we understand the complexities of discovery, the challenges of development, and the critical importance of quality and compliance. Explore our comprehensive solutions, designed to support you at every stage—from pioneering discovery technologies to robust QA/QC protocols and advanced manufacturing systems. Partner with us to navigate the evolving therapeutic landscape.

Discover how we can empower your research journey at ESGCT.

Meet us at booth #A04

For research use only. Not for use in diagnostic procedures.

What's New

Accelerate research into reality

At ESGCT 2024, Revvity will showcase its latest innovations designed to empower breakthroughs in cell and gene therapy. Here are some of the cutting-edge technologies you'll discover at our booth:

  • Pin-point™ Base Editing Platform
    Experience efficient gene editing with our Pin-point™ base editing platform. This modular system enables simultaneous knock-in and multiplex knockout, optimized for T-cells and iPSCs
  • Viral Vector Engineering and Manufacture 
    Optimize your gene therapy production with our AAV and lentivirus engineering solutions. Our custom viral vector development services help you enhance delivery system efficiency, while ensuring manufacturing success through process optimization​.
  • LentiBOOST™ Lentiviral Transduction Reagent
    Used in over 40 clinical trials, LentiBOOST™ increases lentiviral transduction efficiency across various cell types, offering GMP and RUO reagents for your clinical and research needs.
  • IVIS™ Spectrum 2 Optical Imaging Platform
    Non-invasive IVIS™ Spectrum 2 provides reliable optical imaging for tracking disease progression and understanding biological changes. This tool supports longitudinal imaging, helping guide drug development​.
  • Cellometer Ascend™ Automatic Cell Counter
    The new Cellometer Ascend™ offers rapid cell counting and viability analysis with autofocus, dual fluorescence, and brightfield imaging, all delivered in under one minute.
  • EnVision™ Nexus™ Multimode Plate Reader
    For high-throughput screening, the EnVision™ Nexus™ provides advanced detection technologies, combining speed and reliability for your most demanding assays​.
  • Multiomics and TotalSeq™ Reagents
    Uncover the full complexity of cellular biology with Multiomics and TotalSeq™ Reagents. These tools allow for single-cell RNA and protein detection, bulk RNA sequencing, and seamless integration into your discovery workflows​.
  • LabChip™ Microfluidic Platform
    Revolutionize your bioanalytical workflows with LabChip™, offering multi-parametric AAV characterization for higher throughput and consistent performance. Get insights into capsid quantity, purity, and ssDNA integrity, all critical for gene therapy development​.

Follow product link for appropriate product disclaimers.

Featured Solutions

Accelerating the power of precision

Discovery – From Identification to Lead Optimization

Navigate the future of technology with comprehensive solutions that help guide your research to new frontiers

Target identification and validation:

Understanding Cell Biology

Discovery Gene Editing and Modulation

Discovery – From Identification to Lead Optimization

Navigate the future of technology with comprehensive solutions that help guide your research to new frontiers

Target identification and validation:

Understanding Cell Biology

Discovery Gene Editing and Modulation

Therapeutic Payload and Delivery

Navigate the course of your therapeutic development with our comprehensive solutions.

Therapeutic Payload

Delivery

Navigate the course of your therapeutic development with our comprehensive solutions.

Therapeutic Payload

Delivery

Guide Pre clinical Success with future-focused Tools

Guide your therapeutic candidates through pre-clinical stages with foresight and precision, setting a clear course for clinical trials.

Preclinical Functional and CQA assays

Imaging and Cell Analysis Systems

Guide your therapeutic candidates through pre-clinical stages with foresight and precision, setting a clear course for clinical trials.

Preclinical Functional and CQA assays

Imaging and Cell Analysis Systems

Scale-up and manufacturing

Streamline your path to scale-up with our solutions and technical expertise, enabling to meet your critical quality attributes for manufacturing success.

Integrated automation systems

Lentivirus Viral Vectors

LentiBOOST™ Technology

Microfluidic Protein Characterization

Streamline your path to scale-up with our solutions and technical expertise, enabling to meet your critical quality attributes for manufacturing success.

Integrated automation systems

Lentivirus Viral Vectors

LentiBOOST™ Technology

Microfluidic Protein Characterization

Scientific Program

Join our thought leaders as they present advancements in our scientific program.

Poster: P0623 - Optimization of the modular Pin-pointTM base editing platform for an engineered Type V CRISPR-Cas effector

Session Date/Time: Thursday 24 October from 14:00 to 15:30

Session Title: Poster Session III

Presentation Room: Exhibit hall

Abstract: The recent appreciation of the variable function of the natural diversity of CRISPR-associated nucleases has led to an interest in expanding the gene editing toolbox. The type V CRISPR-Cas system is particularly attractive for clinical development due to its smaller size than SpCas9 and its unique PAM specificities, permitting a greater variety of delivery vehicles and allowing for enhanced accessibility of editing sites not always attainable with type II CRISPR-Cas enzymes, such as Cas9. Revvity’s modular Pin-point™ base editing platform employs the delivery of either a nickase or deactivated Cas enzyme, a deaminase fused to an aptamer binding protein, and an aptameric guide RNA (gRNA) that assemble in-vivo and act in conjunction to facilitate precise single nucleotide conversions. While originally developed using Cas9 nickase, the modularity inherent to the Pin-point platform permits swapping of components to achieve optimal editing results. Here, we demonstrate robust site-specific editing at different target loci using a deactivated CasONYX (dCasONYX) version of the Pin-point base editing platform. dCasONYX is an engineered version of dCasMINI developed by Epic Bio. It has an improved off-target profile compared to SpCas9, exhibits very low immunogenicity compared to Cas9 and is less than 1.5 Kb in size. Further utilizing the modularity of the Pin-point base editing platform, we show that additional components of the system such as different deaminases and aptameric gRNA scaffold configurations can be leveraged in combination with alternative Cas enzymes to adjust the base editing window; this is of particular advantage for applications including SNP correction and gene knockout at sites that are not usually accessible with the nCas9 configuration. Finally, we have significantly improved the design and optimized the delivery parameters of the dCasONYX version of the Pin-point platform through generation of IVT mRNA and achieving robust chemical synthesis of gRNA over 150nt in length, making this configuration suitable for application to therapeutically relevant cell types such as T cells and induced pluripotent stem cells (iPSCs).

 

Poster: P0625 - A single-step process for engineering hypoimmunogenic pluripotent stem cells with the Pin-pointTM base editing platform

Session Date/Time: Thursday 24 October from 14:00 to 15:30

Session Title: Poster Session III

Presentation Room: Exhibit hall

Abstract: Pluripotent stem cells (PSCs) hold great promise for the manufacturing of numerous advanced cell therapies. Off-the-shelf allogeneic products derived from PSCs engineered to be compatible with large cohorts of patients have the potential to dramatically broaden access to these therapies, however their sensitivity to DNA damage presents challenges for efficiently performing the complex genome editing operations necessary to realise much of their potential. Base editors represent a potential solution to these challenges due to their reduced genotoxicity compared to nuclease-based technologies. We have developed the Pin-point™ platform, which enables the modular assembly of base editors composed of DNA binding Cas and DNA modifying deaminase components associated via an aptamer encoded in the sequence-targeting guide RNA (gRNA). Owing to the aptamer-dependent recruitment of the deaminase component to target DNA sequences, the Pin-point platform uniquely allows multi-purposing of a single Cas nickase component for simultaneous multiplexed base editing and targeted transgene knock-in. Transient delivery of mRNAs encoding a Pin-point base editor composed of Rat APOBEC1 and SpCas9 nickase in combination with synthetic aptamer-encoding gRNAs achieved durable target protein knockout, and substantially improved cell viability, editing efficiency, and genome integrity following multiplexed base editing compared to CRISPR-Cas9 with no adverse impacts on pluripotency. To demonstrate the utility of the Pin-point platform for the engineering of allogeneic PSCs we generated a panel of clonal hypoimmunogenic iPSC lines with a range of genotypes using an automated clone tracking and picking workflow. Hypoimmunogenic iPSC lines generated via both multiplexed base editing and simultaneous base editing with targeted transgene integration retained pluripotency and exhibited the expected human leukocyte antigen (HLA) phenotypes when differentiated to therapeutic cell products. The Pin-point platform therefore represents a safe and efficient solution to simultaneously perform multiple genome engineering operations via a novel single step process compatible with downstream automation, offering the opportunity to dramatically streamline the development of allogeneic iPSC-derived cell therapies.
 

Poster: P0667 - Interchanging Cas enzymes, deaminases, and aptamer-guide RNA combinations to achieve optimal editing with the modular Pin-pointTM base editing platform

Session Date/Time: Thursday 24 October from 14:00 to 15:30

Session Title: Poster Session III

Presentation Room: Exhibit hall

Abstract: Base editing was first described in 2016 as a powerful tool to introduce precise genomic changes by avoiding DNA double-strand breaks and it has rapidly progressed to the clinic. However, its original Cas9 configuration is not able to address all genetic changes due to PAM limitations and the available deaminases. The uniquely modular Pin-point base editing platform is a three-component system consisting of either a nuclease-deficient or nickase Cas enzyme, plus an extended guide RNA with an aptameric scaffold, and an aptamer binding protein fused to a deaminase. These three components can be efficiently delivered as mRNA and synthetic sgRNA in primary T cells, iPSCs, and HSPCs to efficiently edit DNA targets of interest. Using the basic configuration of the system with a nickase S. pyogenes Cas9 (nSpCas9) and rat-APOBEC deaminase, the targeting capacity for base editing is limited due to the NGG PAM requirement of the nSpCas9 enzyme along with an editing window primarily targeting positions 4-8 within the protospacer for the rat-APOBEC deaminase. To target additional genomic locations, we have leveraged the modular nature of the Pin-point platform for an easily adapted “plug-and-play” approach in which we introduce different combinations of nucleases, deaminases, and sgRNAs containing varying aptamer positions within the gRNA scaffold . We have developed an arrayed screening platform for high-throughput characterization of these additional configurations as a way to demonstrate the unique editing characteristics of these novel systems. Using alternative Cas enzymes such as members of the Cas12 family and its T-rich PAM requirements enables editing at target sites that are not readily addressable using Cas9. Additionally, use of different species of deaminases such as the lizard derived Anolis-APOBEC enables editing with a wider editing window. When used in combination with different aptamer binding proteins and sgRNA scaffolds, we show that regions of the genome not previously attainable with previous configurations can now be efficiently edited . We also show that when using a dual aptameric approach, simultaneous editing with multiple deaminases can be performed in a single transfection as a way to edit two or more target sites and without the need for introduction of orthogonal Cas enzymes to deliver the different deaminases. This modular approach to base editing enables highly specific and efficient editing that can be custom tailored to fit specific cell and gene therapy programs and their unique editing requirements.

Poster: P0669 - Precise and efficient immune cell engineering via Pin-pointTM base editing platform: insights from TotalSeq single-cell multiomic Analysis

Session Date/Time: Thursday 24 October from 14:00 to 15:30

Session Title: Poster Session III

Presentation Room: Exhibit hall

Abstract: Many gene editing platforms, such as CRISPR-Cas9, rely on nuclease activity to introduce a targeted protein knockout through the introduction of DNA double-strand breaks (DSBs). While this revolutionary platform has been used in a wide array of cell and gene therapy applications and at varying stages of pre-clinical research up through recent FDA approvals, the technology does not come without risks. DSBs introduced by nucleases can lead to unwanted genomic alterations and cytotoxicity. These effects are magnified when more complex genomic perturbations, such as editing at multiple genomic sites, are introduced. Here, we demonstrate efficient multiplex gene editing using the Pin-point base editing platform as a safe and suitable technology for the creation of CAR-T cell therapies. The Pin-point platform can efficiently knockout four or more protein targets simultaneously with limited impact on cell viability in T cells and can also be utilized for a number of iPSC and HSPC therapeutic engineering applications. Through single-cell multiomic approaches, we can characterize the effect of editing with the Pin-point platform at the RNA and protein level. We see that the platform can efficiently edit multiple subpopulations of cells, for example, CD4, CD8, and dual CD4/CD8 positive T-cells, with high on-target efficacy and with no detectable off-target phenotypic impacts identified via interrogation of the whole transcriptome and a highly-multiplexed protein panel. From discovery to pre-clinical development, the Pin-point and TotalSeq platforms are well suited for engineering and characterizing next generation cell and gene therapies.

Poster: P0673 - Minimal activation of the p53 DNA damage response by a modular cytosine base editor enables effective multiplexed gene knockout in induced pluripotent stem cells

Session Date/Time: Thursday 24 October from 14:00 to 15:30

Session Title: Poster Session III

Presentation Room: Exhibit hall

Abstract: Precise genome editing of induced pluripotent stem cells (iPSC) holds great promise for engineering advanced cell therapies. CRISPR-Cas systems have been widely adopted in genome engineering applications, however their dependence on genotoxic DNA double strand breaks (DSBs) presents challenges in hypersensitive iPSCs, including the selection for defective DNA DSB responses. Base editors are capable of both modifying and ablating gene function without generating DSBs making them an attractive solution in iPSC engineering applications. Here we report efficient and durable target knockout and substantially improved cell viability and expansion with a cytosine base editor assembled using the Pin-point™ platform compared to SpCas9. The cytosine base editor minimally activated p53-mediated DNA damage signalling independently of the number of simultaneous edits installed. By contrast, multiplexed editing with SpCas9 lead to high levels of p53 signalling and an associated reduction in editing efficiency. While transient inhibition of the p53 DNA DSB response enhanced SpCas9 multiplexed gene editing efficiency this was not required to achieve maximal base editing efficiency. Multiplexed editing of iPSCs with Pin-point base editors therefore both enhances the efficiency of genome engineering processes and substantially reduces the risk of selection for defective DNA damage responses inherent to DSB-dependent CRISPR-Cas systems.

Poster: P0767 - One-step generation of allogeneic CAR-T cells by simultaneous multiplex knockout and site-specific transgene integration with the Pin-point™ base editing platform

Session Date/Time: Thursday 24 October from 14:00 to 15:30

Session Title: Poster Session III

Presentation Room: Exhibit hall

Abstract: As our understanding of immune function regulators and the effects of the tumour microenvironment on immune cells improves, increasingly more complex cell engineering is being employed to improve the efficacy of cellular immunotherapies. We have developed the RNA aptamer-mediated Pin-point base editing platform with the aim to facilitate this complex cell therapy engineering in a safer manner than conventional nuclease-based technologies. The Pin-point base editing system is a modular technology where the CRISPR-Cas and the deaminase modules are delivered to the target cells as individual components. The assembly of the base editing machinery at the target locus relies on the interaction between an aptamer binding protein fused to the deaminase and an RNA aptamer located on the gRNA. The modularity and aptamer-dependent nature of the technology supports high flexibility in the customization of each individual component to address specific editing needs and enables complex genetic modifications. In this example, by combining aptamer-containing and aptamer-less gRNAs, we generated functional engineered CAR-T cells via simultaneous knockout of multiple targets by base editing alongside targeted chimeric antigen receptor (CAR) insertion at the endogenous TRAC locus. We used aptamer-less gRNAs to direct the nickase activity of the Cas enzyme to both DNA strands of the integration locus and stimulate homologous dependent repair (HDR) but avoiding the recruitment of the deaminase; whilst in the same transfection, aptamer-containing gRNAs recruit the deaminase to the other target sites for knockout generation by base editing. With this approach, site-specific knock-in and multiplex gene knockout are achieved within a single intervention and without the requirement to deliver additional sequence-targeting components, the introduction of multiple nuclease species, or more convoluted sequential editing strategies. We demonstrated high base editing efficiency and confirmed the safety of this approach by assessing the editing purity at all target sites, and carefully characterising the occurrence of DNA and RNA off targets, as well as genomic structural variants. The modularity and aptamer-dependent nature of the Pin-point base editing technology opens the possibility of specifically optimizing editing for each site in a different way and of combining multiple effectors to achieve advanced editing outcomes, broadening the applicability of this editing approach across oncology, autoimmunity, and the treatment of rare disease. both enhances the efficiency of genome engineering processes and substantially reduces the risk of selection for defective DNA damage responses inherent to DSB-dependent CRISPR-Cas systems.

Poster: P0530 - Optimization of the modular Pin-pointTM base editing platform with an AI-engineered CRISPR-Cas effector

Session Date/Time: Thursday 24 October from 18:00 to 19:30

Session Title: Poster Session IV

Presentation Room: Exhibit hall

Abstract: The application of language models and artificial intelligence (AI) in protein design represents a transformative approach in the discovery of novel proteins. This approach offers significant promise, particularly in the gene editing field where naturally occurring enzymes that work well in mammalian systems are limited. Traditional methods of protein engineering often rely on labour-intensive and time-consuming processes such as random mutagenesis, DNA shuffling and directed evolution. In contrast, AI-driven models, inspired by natural language processing techniques, can enable the generation of novel proteins with optimal functional properties bypassing evolutionary constraints. Profluent’s OpenCRISPR-1 is the first AI-generated gene editor that has been released to the market, demonstrating editing capacities similar to those of SpCas9.

Base editors were first described in 2016 as a promising next generation genome editing tool that has rapidly progressed to clinical applications. Revvity’s Pin-point base editing platform is a modular system which in some configurations consists of either a nuclease-deficient or nickase Cas enzyme, an aptameric guide RNA (sgRNA), and a deaminase fused to an aptamer binding protein. These components assemble within the cell and act in concert to facilitate precise single-nucleotide conversions. The inherent modularity of the Pin-point platform allows for the swapping of nuclease and deaminase components to achieve optimal editing results. Here, we demonstrate robust editing by incorporating a nickase OpenCRISPR-1 enzyme into the Pin-point base editing platform. The Pin-point editing machinery in this example is tailored for base editing applications to therapeutically relevant cell types such as T cells and induced pluripotent stem cells (iPSCs). Using base editing to induce gene knockout, we targeted four therapeutically relevant genes for the development of allogeneic CAR-T cells, achieving high editing efficiency at all targeted sites simultaneously. The modular nature of the Pin-point system offers the unique advantage of enabling simultaneous site-specific knock-in of a CAR using aptamer-less gRNA while performing base editing on other sites using aptamer-containing gRNAs limiting the drawbacks of sequential delivery, gRNA crosstalk, or the requirement of orthogonal Cas enzymes. By having successfully integrated the open-access nuclease OpenCRISPR-1 with our innovative Pin-point base editing platform, we demonstrate the versatility of our platform and provide further choices when applied to advanced gene editing technologies. This combination shows high levels of base editing precision and efficiency in a therapeutic context and exemplifies a significant leap forward in the development of tools for cutting-edge genomic medicine.

Poster: P0628 - Highly efficient base editing of human hematopoietic stem and progenitor cells with the Pin-pointTM platform

Session Date/Time: Thursday 24 October from 18:00 to 19:30

Session Title: Poster Session IV

Presentation Room: Exhibit hall

Abstract: Hematopoietic stem and progenitor cells (HSPCs) are a foundational cell type for the development of engineered therapies. Given their susceptibility to DNA damage, it is crucial to employ gene editing technologies that minimize genotoxicity. Base editors, such as our Pin-pointTM platform efficiently mitigate the challenges posed by nuclease-induced double-strand breaks (DSBs), such as activation of the DNA damage response and chromosomal aberrations. Our Pin-pointTM platform is a modular base editor allowing ratio optimisation and switching out of the components for different applications. It is capable of complex genetic modifications in a single intervention without relying on the introduction of DSBs as we have shown in primary human T cells and iPSCs. The advanced safety profile of this technology makes it well suited to sensitive cell types such as HSPCs. In HSPCs we used a Pin-point base editor composed of Rat APOBEC1 and SpCas9 nickase mRNAs to achieve up to 80% C to T conversion at the B2M locus with high levels of editing purity and very low incidence of indels, while retaining the most primitive HSC population. We also targeted two separate loci known to reactivate γ-globin expression and achieved a high level of base editing at both loci that corresponded with an increase in γ-globin mRNA and protein expression. Edited HSPCs retained viability, immunophenotype, and differentiation potential toward the erythroid lineage in vitro. The ability to base edit HSPCs efficiently and safely, while retaining high cell viability and differentiation capability, demonstrates the strength of the Pin-point platform as a tool for the generation of advanced cell therapies using sensitive cell types.

Poster: P0646 - Development of a Novel SMARTvectorTM Multiplex shRNA Platform for Safer Cell Therapy Engineering

Session Date/Time: Thursday 24 October from 18:00 to 19:30

Session Title: Poster Session IV

Presentation Room: Exhibit hall

Abstract: Chimeric antigen receptor (CAR) T cell therapy represents a new era of cell-based immunotherapies, allowing for targeted killing of cancer, often blood cancers, expressing a specific antigen. Despite the success of several CAR T cell therapies achieving FDA approval, these therapies often suffer from T cell exhaustion, which can present as reduced cytolytic activity, increased expression of inhibitory receptors, and reduced proliferative capacity. These outcomes render the treatment less potent, pointing towards a need to develop more advanced and tunable methods of modulating CAR T cells to be more robust and reliable. Gene editing approaches such as CRISPR-Cas9 have successfully been used for disruption of genes such as PDCD1, an inhibitory receptor involved in CAR T cell dysfunction, to enhance CAR T cell performance in vitro. Despite the efficiency of CRISPR-Cas9, the number of targets that can be edited in a single sample is partially limited by increased cytotoxicity associated with DNA double-strand breaks (DSBs), thus limiting the therapeutic applicability. Here we present the SMARTvector lentiviral shRNA technology, a method for simultaneous multiplex gene knockdown in immune cells from delivery of a single expression vector. We show that up to eight shRNAs and an anti-CD19 CAR can be expressed after a single lentiviral transduction, thus providing a safer alternative of targeting multiple genes while maximizing CAR T cell engineering.

We developed the SMARTvector multiplex shRNA technology with multiple repeats of a novel patented microRNA scaffold, flanking artificial DNA sequences that target mRNA transcript(s) of interest for efficient gene knockdown. The vector design includes a tunable promoter and selection markers for specific targeted development. Here, an array of multiple shRNAs was generated in a single expression vector with efficacy measured by RT-qPCR to assess relative mRNA transcript knockdown or flow cytometry for assessment of functional protein knockdown. We have demonstrated that multiple shRNAs can be expressed from a single expression vector, resulting in sustained expression and efficient gene knockdown in primary T cells and iPSCs. In addition to knockdown efficiency, we have engineered a vector with optimal linker length between each microRNA-based shRNA encoding region and evaluated position related impacts of each shRNA on the multiplexed cassette. We demonstrate that the SMARTvector multiplex shRNA technology is highly modular and adaptable, making it primed for use in a wide range of cell therapy applications, such as generation of allogenic CAR T cell therapies.

Poster: P0668 - Development and validation of customized guide RNA design and efficiency prediction tools for the Pin-point™ base editing platform

Session Date/Time: Thursday 24 October from 18:00 to 19:30

Session Title: Poster Session IV

Presentation Room: Exhibit hall

Abstract: Base editors are a class of promising next generation genome editing technologies with the potential to both precisely correct disease-causing genetic variants and to safely knockout multiple gene targets simultaneously. The Pin-point base editing platform is a modular assembly of DNA binding Cas and DNA modifying deaminase components associated via an aptamer encoded in the sequence-targeting guide RNA (gRNA). A major challenge in the application of base editors in general is accurately in silico predicting the efficiency and specificity of editing at target sequences for a given combination of Cas and deaminase components to create a shortlist of possible gRNA designs for experimental validation. The modularity of the Pin-point base editing system allows the creation of a large number of configurations, that can vary in their PAM specificity, sequence editing preference and editing efficiency. To facilitate and accelerate the development of applications based on the Pin-point platform, we created a custom tool to design gRNAs to target the gene of interest and to install base conversions, including those that would either install premature STOP codons or destroy splice sites to knockout the target gene. Additionally, we performed a massive parallel cell-based screen to analyze the editing activity of two different Pin-point base editor configurations with gRNAs targeting thousands of target sequences, including 7009 pathogenic SNVs from the ClinVAR database. We used the data obtained from the screen to construct models of the observed editing outcomes for each configuration. We applied these models to rank gRNAs designed to generate functional knockout at multiple clinically-relevant gene targets, including CIITA and PCSK9. After analyzing the correlation of the in silico prediction with the cell-based performance of the gRNAs, we confirmed that the model predictions accurately correlate with the observed editing efficiency for the Pin-point base editing platform. The predictive guide RNA design tool led to the identification of a novel highly efficient gRNA able to knockout PCSK9 by disrupting a splice site, and we confirmed the predicted performance of other gRNA designs previously reported in the literature. Our gRNA design rules were informed using our broad cell-based performance dataset, creating reliable custom tools to design gRNAs and select those with the highest editing efficiency.

Poster: P0186 - Design of experiments in rAAV manufacturing: Optimization of plasmid ratio for better upstream yield

Session Title: Poster

Abstract: Recombinant adeno-associated viruses (rAAV) are very popular for gene delivery by virtue of their tissue specificity, low pathogenicity, and long-term efficacy. Since the FDA approval of Luxturna in 2017, six more AAV-based therapies have entered the global market including Beqvez™ for the treatment of haemophilia B. The one-time treatment for severe haemophilia B either with Hemgenix® or Beqvez™ has a price tag in the US of $3.5 million per dose. One of the most expensive raw materials for making rAAVs by transient transfection are the plasmids, that code for the different parts of the virus including the outer capsid. Identifying the optimal amounts of plasmids needed to produce the most, functional rAAVs – albeit from a seemingly endless number of possible combinations – is key to driving down the cost of these therapies. Design of experiments (DoE) is a statistical approach to identify how multiple factors affect a response, and reduce the number of conditions, while still maintaining statistical power. To optimize the production of rAAVs, we reduced the number of 3-plasmid ratio combinations needed to be tested from 14553 to 28, by generating an optimal design using an R-based open-source DoE pipeline. A HEK293F-derived clonal suspension cell line was transfected using the 3-plasmid system in vented 50 mL tubes with 10 mL of cell culture. Cells were harvested 70 hours later by surfactant lysis and endonuclease digestion then clarified by centrifugation. The clarified cell lysate was measured by ITR2-qPCR. We identified conditions of total plasmid DNA along with the optimal ratio of pHelper to pRepCap to pTransgene which had up to 4x higher productivity than others. The results were used to create a Response Surface Model (RSM), which was confirmed by testing the values suggested by ridge analysis, followed by a comparison of our previously best-performing plasmid ratio against the ratio suggested by the RSM. An increase of up to 3x more vector genome titres across four common serotypes and two transgenes of different lengths was found, with productivity for some serotypes at >7E+14 VG/L of clarified cell culture. The method can be applied at the pre-discovery stage to identify capsids and transgenes with better productivity, or later during the preclinical stage to fine tune the plasmid amount for better empty:full ratios.

Poster: P0692- Developing image-based cell assays to effectively monitor T-cell apoptosis and CAR expression .

Session Date/Time: Thursday 24 October from 18:00 to 19:30

Session Title: Poster Session IV

Presentation Room:

Abstract: Chimeric antigen receptor (CAR)-T cell therapy is a novel cellular therapeutic approach for cancer patients, including B-cell malignancies. A critical step in the CAR-T cell production process is to effectively deliver CAR genes into primary T- cells, which can be achieved through the use of viral vectors or non-viral methods. To evaluate and select effective CAR gene delivery methods and processes, it is imperative to perform analytical tests to detect and monitor cell proliferation, cell health status, and CAR gene expression. In this work, we developed an image-based method using the Cellaca® PLX Image Cytometer to quickly count T-cells, measure viability, assess apoptotic cell health, and identify CAR expression. Using this new methodology, we compared different CAR gene delivery methods, primarily focusing on non-viral methods involving electroporation. Cell viabilities were monitored daily using acridine orange / propidium iodide (AO/PI) stain and its respective dual fluorescent assay. Preliminary results showed that viabilities for all SupT1 samples decreased significantly to ~50% by day 1 following electroporation, in comparison to un-transduced SupT1 control samples, which maintained ~90%+ viabilities. These results confirmed that the introduction of plasmids, rather than the electroporation process itself, induced apoptosis and eventually cell death. Additionally, Annexin V / PI and Caspase-3 / RubyDead cell health assays were tested, and results indicated that a majority of cell death following electroporation was likely the results of apoptotic cells transitioning to the point of no return – cell death. Transduced SupT1 samples were able to fully recover, as their viabilities increased to ~90%+ by day 5 of the study. Lastly, samples were stained with APC-conjugated specific anti-CAR antibody, and SupT1 CAR expression levels were measured using the Cellaca® PLX, and results were confirmed using a flow cytometer. Utilizing this image-based method, we were able to monitor CAR expression in SupT1 cell samples on day 2, 5, 7 and compare CAR expression levels among different gene delivery methods (viral vectors or non-viral methods). With the advantages of ease of use, visual verification with captured cell images, and higher-throughput capability, the Cellaca® PLX Image Cytometer may be potentially used as a convenient benchtop system for rapid assessment of the quantity and quality of CAR-T cells, which may ultimately improve the productivity of development and manufacturability of CAR-T products.