Omics: Unlocking a New Era of Quality Control and Treatment Insight for Cell and Gene Therapy

With omics technologies, leaders in biomanufacturing innovation can provide the next level of insight into safety and efficacy of cell and gene therapies. Exactly what therapy makers are looking for.

The field of cell and gene (C&G) therapy has made remarkable strides, revolutionising the treatment landscape for monogenic diseases and B-cell leukaemias and lymphomas, but the journey ahead demands caution. These therapies, though promising, are complex, posing challenges to ensure consistent quality.

While most advanced therapies cater to high-risk patient populations, where the benefits typically outweigh the risks, expanding their reach to complex conditions like diabetes necessitates deeper interventions and improved quality control (QC) strategies.  

Vigorous QC measures must be implemented at every stage, including establishing robust manufacturing processes, characterising starting materials, and monitoring and testing of the final product for sterility, purity, potency, and efficacy. 

Omics technologies are emerging as a promising solution to address many of these challenges, by providing a comprehensive understanding of target tissues, sterility and purity of the final product, therapeutic effectiveness, and disease-specific interactions.  

At TTP, we have a keen interest in the challenges of C&G therapy and the potential role of omics in addressing them. As a company with expertise from fundamental biology to robotic automation, it is no wonder that this field excites us.  

The urgency of patient needs, and the limited shelf life of C&G therapies often pose challenges for release quality control sterility testing. Conventional pharmacopeial methods, including USP <71>, demand large sample volumes and lengthy incubation periods, often up to 14 days.  

In the case of autologous product processes, where each patient sample is treated as a single batch, there is insufficient time or material to conduct traditional pharmacopeial sterility testing. Furthermore, it is often necessary to detect a needle in a haystack – a single microbe amidst millions of T-cells.

To overcome these challenges, innovative approaches tailored for C&G therapy products are needed. Omics technologies, including metagenomics and genomics, provide promising solutions by enabling comprehensive analysis of microbial DNA, allowing for the high-sensitivity identification of potential contaminants through amplification and sequencing the entire DNA content in a sample.

These techniques offer the potential to expedite the detection of microbial contaminants in shorter timeframes, aligning with the demands of time-sensitive cellular therapies. At TTP, we have partnered with clients to developed isothermal amplification-based solutions that precisely addresses these requirements.

But sterility testing is just one step in the process to a safe and efficacious therapy.

The recognition of disease heterogeneity and variable therapeutic responses has become widely accepted, and C&G therapies are no exception. Therefore, it is essential to establish safeguards not only to ensure safety, but also to predict the efficacy C&G therapies in individual patients. 

The development of a C&G therapy involves addressing numerous critical questions to assess effectiveness. These may include assessing viral transduction rates for vector-based therapies, assessing heterogeneity in on- and off-target editing by CRISPR-based approaches as well as aberrant translocations, and considerations of zygosity. C&G therapies used as therapeutics can exhibit heterogeneity in their genetic alterations, lineage, or state. Such variations have the potential to impact both the efficacy and safety of these products. 

Traditional bulk sequencing methods may mask crucial factors. For example, conventional PCR analyses typically provide an average copy number for a given batch, potentially overlooking cells with excessively high copy numbers. In addition, PCR-based QC approaches do not identify architectural changes within and around the integration sites, copies maybe inverted and/or truncated. To gain a comprehensive understanding, it is useful to measure the distribution of vector copy numbers at the individual cell level.  

Single-cell multi-omics approaches provide the necessary resolution to unravel complex systems and offer a unique opportunity to characterise novel C&G therapies. Examining individual cells at the genomic, transcriptomic, and/or proteomic levels can identify cellular heterogeneity, characterize cell populations responding to the therapy, and assess the overall therapeutic effect at a single-cell resolution. 

For example, Mission bio has developed the Tapestri platform, a novel two-step microfluidic workflow to access DNA and protein in single cells. By leveraging single-cell multi-omics technologies, copy number, small nucleotide variants and functional proteins at a per-cell level can be assessed providing a deeper understanding of cell potency and efficacy. 

PhenomeX offers a platform that effectively captures cytokine, chemokine, and phosphoprotein signatures from individual cells. This platform enables the prediction of disease progression, treatment resistance, and therapeutic efficacy even before infusion of the product into the patient. 

Once gene-corrected cells are infused into patients undergoing gene therapy, it becomes critical to monitor the clonal evolution of these corrected cells. Single-cell whole-genome sequencing (scWGS) can be utilised to track these dynamics and provide insights into whether treatment-related mutations occur during the therapy process.  

Similarly, studies employing single-cell RNA sequencing (scRNAseq) examine cell populations before and after infusion, leading to clinically relevant conclusions regarding transcriptional profiles associated with favourable or unfavourable clinical outcomes. 

Long-read sequencing and genome mapping technologies also present a promising approach for the QC of C&G therapy products. Two sequencing technologies currently dominate the long-read sequencing space: Pacific Biosciences’ (PacBio) single-molecule real-time (SMRT) sequencing and Oxford Nanopore Technologies’ (ONT) nanopore sequencing.  

These advanced sequencing methods offer several key advantages:  

Firstly, they enable comprehensive architectural analysis, allowing for the accurate identification of the therapeutic sequence and its integration site within the host genome. The ability of long-read sequencing to reconstruct structural variations in the genome, provides insight into potential genomic rearrangements that could impact the therapy’s effectiveness or safety. For example, the optical genome mapping technology from Bionano’s Saphyr is performed on ultra-high molecular weight DNA, ensuring that structural variants are observed and not inferred as with traditional NGS.  

These technologies can also facilitate the characterisation of viral vectors used in gene therapy, ensuring their correct viral payload  insertion during manufacture and subsequently tracking structure of the payload once delivered into a host, ensuring episomal DNA has not integrated.  

Furthermore, long-read sequencing allows for a more accurate and comprehensive measurement of transgene expression levels by being able to identify splice variants and fusion genes, providing valuable information about the therapeutic activity of the introduced genes. 

Recently, researchers have performed long-read sequencing in single cells. By taking advantage of droplet-based amplification they generated long reads using PacBio HiFi technology on single human T cells. Compared to short-read sequencing, the new method captured four times as many structural variants.  

By leveraging long-read sequencing in QC processes, C&G therapy producers can obtain a more complete assessment of product quality from manufacture to patient monitoring.

This comprehensive analysis offers the promise of a robust framework for quality assurance and regulatory compliance. 

There remains a common conception among developers that omics technologies are both costly and complex, involving time-sensitive sample handling, multiple procedural steps, and skilled technicians. Furthermore, characterising C&G therapies comprehensively has posed logistical challenges, as it involves harmonising data from various platforms and instruments. Even with considerable efforts, these inefficient ad hoc workflows often fall short in capturing cell-to-cell variation effectively.   

Additionally, despite the recent decrease in the cost of sequencing, this remains heavily reliant on economies of scale. The affordability of sequencing is primarily achieved when performed in large batches, consisting of hundreds to thousands of samples. However, for C&G therapies to capitalise on the speed of omics technologies, they cannot always take advantage of batch sequencing runs since samples need to be analysed timeously in smaller batches.  

Coupling this to the need for skilled laboratory users to carry the majority of sequencing protocols – even more true with the addition of complex enrichment steps for capturing the therapeutic sequence – there is a need for a deskilled sample-to-library instrument enabling HMW DNA extraction, enrichment and library preparation reducing the time and skill level required to carry out the QC.  

At TTP, we have extensive expertise in the development of systems for sample preparation, cell encapsulation, and streamlining library preparation. Our proficiency spans both the automation and fundamental biology aspects of these processes. We have the capability to develop sequencers and sequencing biochemistry from the ground up, all the while improving their user-friendliness and decreasing manufacturing costs. And, we have extensive experience in automating these processes.  

TTP has successfully developed more than 10 cell therapy systems in just 5 years. One of TTP’s spinoffs, Cellular Origins, brings robotic automation to the field of C&G therapy. They enable cell therapy developers to efficiently expand their manufacturing capabilities and gain access to the necessary tools and technologies at the required scale. 

It is within reach to envision custom developments that strive to achieve end-to-end, streamlined solutions for sample and library preparation, as well as sequencing. These advancements have the potential to achieve costs comparable to what is currently attainable only with high-throughput sequencers, but on a smaller scale involving single or a limited number of samples. 

By early investment in omics, pioneers in C&G therapy can gain a competitive edge, positioning themselves at the forefront of scientific advancements, and uncovering hidden insights on safety and efficacy.  

If you require a custom solution that takes advantage of omics for C&G therapy, where it involves single cells or long-read sequencing, together we can develop a tailored, end-to-end solution to meet your specific needs. Join TTP in making omics for QC in C&G therapy a reality.