Background & AimInterest in gene therapy-based disease prevention and treatment has grown rapidly over the last decade. While lentivirus has been the viral vector of choice for gene therapy, recombinant adeno-associated viral (rAAV) vectors have seen recent widespread application due to the non-integrating ability. Baculoviruses are emerging as a highly adaptable vector with a broad tissue and host tropism. Regardless of vector, extensive quality control (QC) throughout the entire development and manufacturing process is essential. A robust QC process expedites safe, effective commercialization of final product. While Sanger sequencing can be ideal to verify and validate sequences pre-packaging, next generation sequencing (NGS) combined with post-viral production methodologies like CE analysis and transmission electron microscopy offer an effective high-throughput approach for monitoring quality, from initial construct assembly to analysis of the encapsulated product. Both short-read and long-read sequencing technologies offer distinct advantages including sequencing of the entire viral genome, with detection of potential mutations, truncations, and contaminants.Methods, Results & ConclusionHere we describe our novel proprietary vector-agnostic workflows, for use in lentiviral, AAV or baculovirus settings. Starting with high quality synthesis of either vector plasmid or region of interest, full-length plasmid sequence is confirmed. Depending on these results, correction or new synthesis of plasmid options are applied. Following packaging within the viral vector system of choice, QC results using both short- and long-read NGS platforms are supported with a regulatory-compliant Sanger assay. State of the art synthesis reduces potential upstream errors. Sanger ITR sequencing and sequence correction upstream alleviates potential downstream issues in viral packaging. High-quality viral packaging ensures robust viral titers for downstream use. The combined NGS approach post-packaging alleviates current constraints for high throughput AAV sequencing and thereby enhance the overall QC process. Our Good Laboratory Practices (GLP) Sanger sequencing method extends read lengths through the entire ITR regions, allowing for rapid sequence confirmation of the final AAV product. The combination of these approaches enables a comprehensive solution, ideal for sequence confirmation of both transfer plasmid and final packaged product for improved viral vector gene therapy manufacturing in advance of FDA or EMA filings.

Background & AimThe omics era has greatly expanded the repertoire of approaches available for researchers and clinicians to unravel the complexity underpinning human health: Next Generation Sequencing (NGS) approaches can characterize genomes, epigenomes, transcriptomes and proteomes. The analyses are critical to assess in individuals both pre- and post-treatment during therapeutic development and early-stage clinical trials. Peripheral blood mononuclear cells (PBMCs) offer a non-invasive approach that, when combined with omics tools, can provide a near holistic view of immune processes across patient cohorts. Meanwhile, Formalin Fixed Paraffin Embedded (FFPE) tissues are a staple in clinical diagnostics and an ideal means to store archival tissue but can be difficult to work with in traditional NGS assays.Methods, Results & ConclusionHere we detail workflows using both fresh and fixed patient samples to rapidly produce a diverse set of multiomics results including genomics, epigenomics, transcriptomics and proteomics. For fresh blood draws, this starts with automated sample handling and processing to ensure high viability and yield of PBMCs, along with simultaneous plasma separation and collection. Samples are then aliquoted and simultaneously processed for whole exome sequencing, single cell RNA sequencing, epigenetic characterization and Olink biomarker analysis. For fixed tissues, FFPE blocks were serially sliced into various FFPE slides, with a single slide H&E stained. Individual slides were then utilized for genome, epigenome, single cell RNA-seq, and digital spatial profiling. Genome information was captured using hybrid capture based approaches followed by deep NGS on an Illumina platform and analyzed for a variety of variants and tumor mutational burden. DNA methylation was detailed using target capture probes targeting DNA methylation sites. Single cell approaches were applied to explore the transcriptome using 10X Genomics scRNAseq kit, and Digital Spatial Profiling (DSP) was done using the NanoString GeoMx® Whole Transcriptome Atlas and immunostaining.With these robust workflows, all these datatypes can be produced within days of fresh or fixed sample receipt using minimal sample amounts. High throughput integrative omics workflows, as described here, drive greater insights in human health, allowing for a rapid combined approach to address the biological questions at hand.

The omics era has greatly expanded the repertoire of approaches available for researchers and clinicians to unravel the complexity underpinning human health: Next Generation Sequencing (NGS) approaches can characterize genomes, epigenomes, transcriptomes, and proteomes. The analyses are critical to assess in individuals both pre- and post-treatment during therapeutic development and early-stage clinical trials. Peripheral blood mononuclear cells (PBMCs) offer a non-invasive approach that, when combined with omics tools, can provide a near holistic view of immune processes across patient cohorts. For fresh blood draws, this starts with automated sample handling and processing to ensure high viability and yield of PBMCs, along with simultaneous plasma separation and collection, which is then aliquoted for downstream analysis. These PBMC and plasma aliquots were then processed such that whole exome sequencing, whole genome methylation sequencing, single cell and bulk RNA sequencing, Olink Explore HT and Seer proteomic analysis, and metabolomic analysis can all be collected from patient blood draw. Integrated analysis across multiple data modalities allows for holistic views of pathways and processes that are highly impacted, with increased statistical significance than any one modality alone. Inclusion of dual proteomics assays provided a greater breadth of downstream functional results, revealing complementarity between assays. While genomics, transcriptomics, and proteomics provide information about genetic and functional potential, inclusion of metabolomics grants deeper phenotypic insights within and across individuals within a patient cohort. Ultimately, this approach reduces the need for repeated patient collections and lowers bio-storage requirements, making it a more patient-friendly and cost-effective solution, while rapidly producing a diverse set of multiomics results and allows for deep exploration of the molecular underpinnings of cancerous tissue. Andrea J. O'Hara, Tom Cohen, Ethan Stancliffe, Adam Richardson, David Corney, Laure Turner, Haythem Latif, Ginger Zhou. Integrated multiomics unlocks holistic phenotypic insights allowing for deep molecular exploration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3691.

Background & AimWhile lentivirus has historically been the viral vector of choice for gene therapy, recombinant adeno-associated viral (rAAV) vectors have seen widespread application as a gene therapy delivery platform in preclinical and clinical studies. Unlike lentiviral vectors, which are fundamentally integrated into the host genome, recombinant AAV largely remains episomal after transduction into the nucleus of the host cells; however, data has shown AAV may integrate into the host genome in a random, non-homologous manner. The frequency of AAV integration has been estimated at 0.1% to 10% in hepatocytes. In consideration of the theoretical risk of tumorigenesis associated with lentiviral and AAV integration in humans, assessment of viral vector integration in in vitro studies, animal models, and patient samples are recommended by FDA for preclinical and clinical studies and long-term follow-up studies (LTFU). AAV integration analysis presents several challenges, including the random nature of AAV integration sites and low expected integration frequency.Methods, Results & ConclusionTo address this, Next Generation Sequencing (NGS) for viral vector integration analysis has been adopted as a highly sensitive assay. Traditional approaches utilize targeted enrichment sequencing with hybrid-capture or linear amplification PCR in combination with short-read sequencing technology. The addition of long-read sequencing offers the advantage of being able to identify chimeric reads with clear sequence structure as genome-insert-genome. Here we describe multiple mixed ISA approaches in lentiviral and AAV studies, with improved sensitivity and specificity for ideal detection of viral vector integration.

Background & Aim Interest in gene therapy-based disease prevention and treatment has grown rapidly over the last decade, with adeno-associated viral vectors (AAV) emerging at the forefront with widespread application due to the non-integrating ability. With safety as the top priority in all therapies, extensive quality control (QC) throughout the entire development and manufacturing process is essential. A robust QC process expedites safe and effective commercialization of the final product. While Sanger sequencing can be ideal to verify and validate plasmid sequences pre-packaging, next generation sequencing (NGS) combined with post-viral production methodologies such as CE analysis and transmission electron microscopy offer an effective high-throughput approach for monitoring AAV quality, from initial construct assembly to analysis of the encapsulated product. Methodology Illumina® short-read, Oxford Nanopore® long-read and PacBio® long-read sequencing technologies each offer distinct advantages including sequencing of the entire viral genome, with detection of potential mutations, truncations, and contaminants. Here we compare each of these applications as a means to assess the purity, clonality and fidelity of the packaged AAV product. Results When evaluating the same sample with Illumina, PacBio and ONT, we show how these methods compare with detection of contaminating alternatively packaged products, detection of point mutations and indels in the ITRs and region of interest, and detection of full-length vs partially packaged genomes. Based off these results, we also evaluate how sample quality and library preparation can influence the conclusions generated and describe when each method can be ideally used within the therapeutic development workflow, starting from early research to late-stage pre-clinical work. Additionally, we consider regulatory assays, with Good Laboratory Practices (GLP) methods being ideal for late-stage development products. Conclusion Ultimately, a combined NGS approach post-packaging enables a comprehensive solution and enhances the overall QC process, ideal for sequence confirmation of both transfer plasmid and final packaged product for improved viral vector gene therapy manufacturing in advance of FDA or EMA filings.

Abstract Formalin Fixed Paraffin Embedded (FFPE) tissues are a staple in clinical diagnostics associated with solid tumor. Next Generation Sequencing (NGS) approaches characterize genomes, epigenomes, transcriptomes, and proteomes from a single sample source. FFPE blocks from several different tumor tissue types were serially sliced into FFPE slides. Individual slides were then utilized to explore the genome, epigenome, single cell RNA-Seq (scRNA-Seq), and Digital Spatial Profiling (DSP). Here we present a comparative analysis of two transcriptomic modalities within one FFPE tissue sample: fixed-cell scRNA-Seq of the transcriptome and digital spatial profiling of the whole transcriptome. Independent analysis of scRNA-Seq and DSP identified common trends as well as distinct differences within the same sample. Comparative analysis within sample between modalities identified statistically significant differences, driven by local tumor microenvironment, but also highlighted commonalities between methods. Cross sample comparisons showed striking differences, as indicated by tumor type. These studies highlight the strengths of each assay individually, as well as the power of a dual-assay approach for comprehensive analysis of the tumor and its microenvironment. Conclusions from this standalone analysis were further aggregated into downstream multiomics analyses, including proteomics analysis, illustrating the expanding landscape of information that can be extracted from FFPE derived tissue and the potential for novel discovery and diagnostic power integrating these complex data types holds. Citation Format: Anthony Moore, Andrea O'Hara, Pranay Vishwanath, Laure Turner, Haythem Latif, Chris Mozdzierz, Ginger Zhou. Comparative analysis of single cell expression and digital spatial profiling in FFPE tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3770.

Background & AimThe use of plasmids is critical in the gene therapy viral vector workflow: plasmids are widely used for creation of the viral vector construct, along with helper and packaging plasmids for final product creation. While the construct insert is universally sequenced during initial design, sequencing of the backbone of the plasmid is not frequently assessed. Sequence errors within the plasmid backbone, helper and packaging plasmids can have major impacts in downstream packaging and viral vector production. Traditional Sanger approaches can be used for fast and frequent characterization, however limitations within the technology necessitate many sequencing reactions to span the full plasmid, with a known reference sequence. Traditional Next Generation Sequencing (NGS) approaches can overcome the need for multiple reactions and a reference sequence but are historically slower and cost-prohibitive.Methods, Results & ConclusionHere we describe a fast and cost-effective sequencing approach for sequencing circular plasmid DNA to generate a highly accurate and annotated full-length consensus sequences without the need for primer design or a known reference sequence. This long-read sequencing method is comparable in cost and time to Sanger sequencing but unlike Sanger sequencing, overcomes common issues within hard to sequence regions like long nucleotide repeats or inverted terminal repeat (ITR) domains of adeno-associated viruses (AAVs). When used effectively, it can be harnessed to characterize gene therapy viral vector plasmid and associated packaging plasmids required throughout the gene therapy development life cycle without delay, ultimately accelerating the viral vector design and development process.

The omics era has greatly expanded the repertoire of approaches available for researchers and clinicians to unravel the complexity behind cancer onset in humans: Next Generation Sequencing (NGS) approaches can characterize genomes, epigenomes, transcriptomes and proteomes of patient samples. Advanced DNA barcoding and automated microfluidics can take this to the next level, enabling multiomic characterization of single cells. Peripheral blood mononuclear cells (PBMCs) offer a window into the immune system that, when combined with these omics tools, can provide an insight into immune cells mediating anti-tumor responses in cancer patients. Here we detail a workflow using a single blood draw to rapidly produce a diverse set of multiomics results including genomics, epigenomics, transcriptomics and proteomics. This starts with automated sample handling and processing of the primary blood draw to ensure high viability and yield of PBMCs, along with simultaneous plasma separation and collection. These samples are then aliquoted and simultaneously processed for automated and semi-automated whole exome sequencing, single-cell RNA sequencing, methylation sequencing and Olink proteomics assay. Germline and somatic mutations can be detected using whole exome or whole genome sequencing with deep coverage, whereas methylation, ATAC or ChIP sequencing can be used for epigenetic characterization of the same sample. While bulk expression offers a high-level transcriptomics profile, single-cell transcriptomics facilitates detection of gene expression changes in each individual cell type, allowing for analysis of rare cell types including circulating tumor cells. Olink proteomic assays can be utilized for both biomarker discovery and validation, with highly targeted or broad-spectrum panels. With this robust workflow and advanced robotics for sample handling and processing to minimize potential batch effects, all these datatypes can be produced within days of primary sample collection using minimal sample amounts. High throughput integrative omics workflows, as described here, are useful in gaining a multidimensional view of cancer and advance immunotherapies by characterizing immune cell modulation in tumor progression, and can be expanded for use in tumor/normal analysis, evaluation of metastases and exploration of tumor microenvironment. Citation Format: Bhagyashree S. Birla, Andrea O'Hara, Elizabeth Louie, Ben Niu, Haythem Latif, Vanessa Tumilasci, Yang Han, Yongjun Fan, David Corney, Pranay Vishwanath, Wei Wang, Alfredo Staffa, Ilaria DeVito, Laure Turner, Chris Mozdzierz, Peter Nowacki, Ginger Zhou. Harnessing the power of multiomics from a single sample to explore tumor heterogeneity and advancing immuno-oncology research. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6611.

The FDA Guidance to Industry on Long Term Follow-Up (LTFU) After Administration of Human Gene Therapy Products states the importance of longitudinal testing of gene products introduced into human subjects. Depending on the delivery mechanism, the therapeutic gene product may or may not integrate into the genome. Of particular interest are gene-product integrations near proto-oncogenes which might lead to malignancies. The FDA LTFU guidance states that recipients of an integrating gene therapy modality should be tracked for 15 years, while those receiving a non-integrating therapy modality should be tracked for 5 years. Therefore, advanced analytical methods are needed to identify, quantify, and track integration events across the genome. Here, we provide a comprehensive evaluation of methods leveraging next-generation sequencing approaches for genome-wide analysis of lentiviral integration events. Our analysis employed well-characterized standards consisting of varying copy number and known integration sites. ​ The approaches we characterized can be bucketed into two major groups: PCR amplification approaches and target capture-based approaches. All methods detected true positives with strong correlation to theoretical integration site dosage levels down to 1% allele frequency. Comparatively, PCR amplification-based approaches have lower data requirement per sample suggesting higher sensitivity, greater molecular capture, and lower limit of detection compared with target enrichment-based approaches. Target enrichment-based approaches can afford the flexibility to capture the integrated vector, which is of interest for characterizing partial integration events. While all methodologies performed well in our study, the choice of assay (or assays) for testing will depend on numerous factors including but not limited to the viral vector system and construct and starting material availability. Citation Format: Jeffrey M. Otto, Yongjun Fan, Guanhui Bao, David Corney, Ben Niu, Qiu Yu, Baursha Khan, Laure Turner, Chris Mozdzierz, Haythem Latif, Ginger Zhou. Benchmarking NGS integration site analysis methods in support of long-term safety monitoring of gene therapy products [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB255.

Abstract The FDA Guidance to Industry on Long Term Follow-Up (LTFU) After Administration of Human Gene Therapy Products states the importance of longitudinal testing of gene products introduced into human subjects. Depending on the delivery mechanism, the therapeutic gene product may or may not integrate into the genome. Of particular interest are gene-product integrations near proto-oncogenes which might lead to malignancies. The FDA LTFU guidance states that recipients of an integrating gene therapy modality should be tracked for 15 years, while those receiving a non-integrating therapy modality should be tracked for 5 years. Therefore, advanced analytical methods are needed to identify, quantify, and track integration events across the genome. Here, we provide a comprehensive evaluation of methods leveraging next-generation sequencing approaches for genome-wide analysis of lentiviral integration events. Our analysis employed well-characterized standards consisting of varying copy number and known integration sites. ​ The approaches we characterized can be bucketed into two major groups: PCR amplification approaches and target capture-based approaches. All methods detected true positives with strong correlation to theoretical integration site dosage levels down to 1% allele frequency. Comparatively, PCR amplification-based approaches have lower data requirement per sample suggesting higher sensitivity, greater molecular capture, and lower limit of detection compared with target enrichment-based approaches. Target enrichment-based approaches can afford the flexibility to capture the integrated vector, which is of interest for characterizing partial integration events. While all methodologies performed well in our study, the choice of assay (or assays) for testing will depend on numerous factors including but not limited to the viral vector system and construct and starting material availability. Citation Format: Jeffrey M. Otto, Yongjun Fan, Guanhui Bao, David Corney, Ben Niu, Qiu Yu, Baursha Khan, Laure Turner, Chris Mozdzierz, Haythem Latif, Ginger Zhou. Benchmarking NGS integration site analysis methods in support of long-term safety monitoring of gene therapy products [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB255.

New oncology therapies are driven by multiple approaches, with a heavy emphasis on small-molecule therapeutics to target cancer cells and provide a therapeutic benefit to cancer patients. These small-molecule compounds can be used as a stand-alone treatment approach or may be combined with other compounds to bring enhanced effects. Compound therapeutic discovery relies heavily on high-throughput screening assays for measurement of phenotypic responses following compound treatment of oncology cell cultures which can range from very targeted assays to wide-ranging unbiased approaches. Standard whole transcriptome RNA-seq is a preferred method for unbiased measurement of phenotypic responses but is often overlooked due to limited scalability and high sample screening costs. To address these challenges, we have developed a high-throughput gene expression (HT-GEx) assay that combines a variety of strategies to enable a low-cost and high-throughput alternative to standard RNA-seq. First, a simplified workflow removes upstream RNA isolation steps and tags transcripts directly from cell lysate. This is achieved by incorporating both a sample barcode and a unique molecular index (UMI) during the reverse transcription reaction. Next, we leverage a 3’ end counting approach to enable a reduction in sequencing depth coverage (i.e. sample cost) without compromising gene detection sensitivity. With these key advances, a high-plex and high-throughput screening assay is achievable at a reduced cost by removing the need to purify RNA, tagging transcripts early in the workflow to allow pooling of samples and reducing sequencing depth. We have combined these high-throughput and cost-saving strategies and present results which confirm HT-GEx offers results on par with standard RNA-seq. Gene detection sensitivity based on number of genes detected per millions of reads is highly similar, with gene detection sensitivity saturated at approximately 2 million reads. Additionally, the number of genes detected between replicates for RNA samples and lysate samples demonstrate strong linear correlation, implying highly reproducible results. Thus, high-throughput gene expression is an ideal method for phenotypic screening in oncology cell lines following stand-alone or combined compound treatments. Citation Format: Andrea O'Hara, Michael Stephens, Ilaria DeVito, Laure Turner, Christopher Mozdzierz, Haythem Latif, Ginger Zhou. High-throughput RNA sequencing directly from cell lysates enables reproducible phenotypic profiling for assessing novel compounds and treatments in cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB130.

Bulk RNA sequencing (RNA-seq) is regularly employed for transcriptional profiling but provides only a snapshot of the average population. Particularly when studying disease, understanding differences in diverse cells is essential thus single-cell resolution is a necessity due to significant cellular heterogeneity. High-throughput droplet- and microfluidic-based approaches for performing single-cell RNA-seq (scRNA-seq) are invaluable techniques for uncovering changes in cells such as differential gene expression and epigenetic states in cancer research. A challenge for scRNA-seq, however, is sample preparation as typically tissues must be processed immediately after collection. We and others have reported alternative approaches to stabilize samples prior to analysis such as cryopreservation of cells and tissues and methanol-based fixation of cells. However, the inclusion of these approaches is still limited in many settings due to the challenging logistics involved in sample collection. To further broaden the ability of researchers to deploy scRNA-seq, we report our experiences from testing a pre-commercial workflow developed by 10x Genomics to resolve these sample management limitations. The use of paraformaldehyde (PFA) to fix samples at the collection site allows samples to be transported to remote laboratories for downstream processing without sacrificing integrity or data quality. This advance has enabled new possibilities for sample accessibility, throughput, and batched analysis in basic and translational research settings. Furthermore, this approach also allows for analysis of transcriptome-wide gene expression at high sensitivity with simultaneous cellular readouts. Here we report a case study comparing the PFA method to standard approaches, and highlight advantages and potential applications previously not possible with reported sample preparation strategies. Citation Format: David Corney, Yang Han, Yu Qiu, Yongjun Fan, Michael Stephens, Andrea O'Hara, Laure Turner, Christopher Mozdzierz, Haythem Latif, Ginger Zhou. Evaluation of a novel single-cell RNA sequencing methodology for use in clinical trial settings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB034.