The cell and gene therapy pipeline has witnessed significant growth in recent years.1 With record funding flowing into this segment, a market worth projected at 13 billion US dollars by 2024, several approved therapies and hundreds of clinical trials underway, the industry is undeniably thriving.2,3 This clearly signals the faith that manufacturers and investors place in these advanced therapy medicine products (ATMPs) as a promising treatment modality.3 However, the sector still faces important roadblocks.1,4 Hurdles are mostly 3-fold: technological, regulatory, and economical.1 These must be fully addressed to (1) secure the safety and efficacy of those potentially revolutionary therapies and (2) ensure that they reach those in need as quickly as possible while keeping costs accessible for patients. Therefore, market entry for cell and gene therapy isn’t as straightforward and major considerations must first collectively be overcome to ride the next big wave.
I/ FDA issues strict genetic QC recommendations for cell and gene therapy manufacturing and testing
Earlier this year, 2 new draft guidances on human gene therapy products incorporating genome editing of human somatic cells and CAR T-cell therapies were released by the U.S. Food and Drug Administration’s (FDA) Center for Biologics Evaluation and Research (CBER). These recommendations primarily touch upon the type of information that should accompany IND application. The checklist includes component design, product manufacture and testing, pre-clinical safety assessment and clinical trial design. 5-8
The first documentation suggests including detailed descriptions of the manufacturing process, testing plan and accompanying analytical procedures. Moreover, it lays out the manufacturing and testing data required for each type of genome editing product. Whether there are sufficient grounds to move product testing to clinical trials will largely depend on the availability of in vitro and in vivo preclinical proof-of-concept data. Indeed, these will help evaluate any potential risks related to the product. Additionally, biodistribution studies can shed light on the extent of editing activity in target and non-target tissues. For clinical studies, regulators want to understand the risks that come with the administered product and therefore, advocate the assessment of genetic consequences resulting from on- and off-target editing. Lastly, clinical trial design should include appropriate patient selection, an efficient and safe approach to administration (e.g. data-based dosing), adequate safety monitoring, appropriate endpoints and long-term safety follow-up (i.e. 15 years post-first administration).5,6
The second document focuses on the development of CAR-T products and zeroes in on CMC, pharmacology and toxicology, clinical study design, and autologous or allogeneic CAR T specific-recommendations. Although these recommendations are applicable to other genetically modified lymphocyte products (e.g. CAR-NK, CAR-TCR), their specialized nature might require additional considerations. Therefore, the FDA urges sponsors to communicate early on with the Office of Tissues and Advanced Therapies (OTAT) in the CBER and seek out for a pre-IND meeting for more guidance (i.e. prior IND submission).5,7,8
It is safe to conclude that the FDA wants sponsors to gather sufficient information over the course of product development and encourage submitting data in a stage-appropriate manner.5 Among others, much emphasis is placed on fully characterizing genetic engineering outcomes. Indeed, sponsors will need to:
- Convincingly validate intentional genetic alterations (e.g. integration site analysis)
- Sidestep (as much as possible) risks for off-target modifications
- Scrutinize whether undesired genetic variations (including large structural variants in and around genes of interest) may have accompanied your editing
- Monitor long-term effects of on- and off-target editing.5,8
II/ Manufacturers in need of improved analytical tools for cell and gene therapy
The advent of editing tools has unquestionably unlocked new therapeutic strategies. However, studies continue to caution the need for careful control strategies to validate desired genetic modification. As a case in point, recent publications have reported large deletions and complex rearrangements following CRISPR-Cas9.10-11 Luckily, scientists are busy working out the mechanisms underpinning those translocations and some have already proposed strategies to reduce their occurence.12 On this note, many will recall that several biotech companies saw their clinical trials coming to a halt last year due to the detection of unexpected rearrangement (i.e. chromosomal abnormalities). Evidently, these events were a hard reality check for the nascent field and reinforced the necessity for thorough genetic characterization of all ATMPs.
Another concern is the highly compressed timelines that researchers typically work with.3 In response to this urgency, the use of automated, high-throughput analytical methods into the manufacturing process will enable more expedited development activities. With a surge in interest to develop streamlined, robust GMP-compliant manufacturing processes, Genetic Engineering & Biotechnology News has published an interesting piece weighing in on the most innovative tools for improved genetic characterization and quality control.13
Comprehensive genetic characterization of heterogeneous cell samples in cell and gene therapy
To date, a growing number of scientific publications underscore the importance of complete genetic QC for viral vector and ATMP manufacturing. As a result, several technologies are often mentioned and used in conjunction for validation.
For instance, Cellectis recently unveiled their novel immune-evasive universal allogeneic CAR T-cells with potential for improved persistence. The remarkable work culminated in a Nature Communications publication, in which they describe their experimental setup for multiplex editing of TRAC and B2M locus via TALEN and adeno-associated viral particles strategy and subsequently, analyzed the specificity of TALEN cleavage and AAV6 matrix insertion.15
In contrast, other labs are exploring avenues that sidestep the need for viruses. There, the work conducted in Prof. Dr. Alexander Marson’s (Director of the Gladstone-UCSF Institute of Genomic Immunology) lab is a prime example. In fact, his team described – back in 2018 – a non-viral genome targeting method that offers a robust molecular “cut and paste” system to rewrite genome sequences in human T cells. To safeguard genetic quality, the researchers verified that the integrated sequences did not bring about any unintended disruptions of other critical genomic sites. 16
IV/ Complete genetic QC to identify integration sites in viral vector producer cell lines
Nevertheless, genetic QC isn’t just relevant for (downstream) immunotherapy products. There is also a strong basis to perform comprehensive genetic characterization QC in more upstream manufacturing stages. In fact, complete genetic QC is highly beneficial for viral vector manufacturing. As an illustration, current manufacturing strategies for lentiviral vector production typically involve transient transfection of HEK293T cells.17 However, the presence of SV40 T antigen-encoding sequences raise safety concerns, as studies have shown that SV40-transformed human cells can promote tumors when administered to nude mice.18,19 In a collaborative study, FDA and NIH researchers therefore sought to knock-out the SV40 T antigen-encoding sequences from HEK293T cells via CRISPR. Cell clones lacking T antigen-encoding sequences were then analyzed by PCR, WGS and TLA.20
Another elegant example of how genetic characterization meaningfully contributes to the upstream and downstream manufacturing processes of CGT products is the publication by Trudy Straetemans (Associate Professor at University Medical Center Utrecht) and prof. dr. Jürgen Kuball (senior author and Legal & Regulatory Affairs Committee (LRAC)chair liaison with European Medicines Agency (EMA), European Commission & Industry) and their colleagues. Given the scarcity of information on precise molecular characterization and possible variations vis-à-vis insert integrity and (vector) copy numbers of different gene therapy medicinal products (GTMPs) during the entire production chain, researchers at UMC Utrecht took it upon themselves to examine their TEG001 insert integrity from vector producer cells until medicinal product. Ultimately, these extensive molecular characterization data completed their clinical study dossier for TEG001, thereby establishing a valuable framework for future GTMPs.21 Thus, adopting improved analytical tools during cell line development helps minimize variation across manufacturing lots.
Concluding remarks: a need to collaborate and synergize capabilities to accelerate time-to-clinic for cell and gene therapies
Over the last 10 years, and even more so during the COVID-19 pandemic, we saw a rapidly evolving biopharma landscape. For one, pharmaceutical companies have increased their reliance on CDMOs for drug development and manufacturing.22 These partnership initiatives should be embraced, as collaboration will only help accelerate therapeutic discovery by facilitating effective transfer of scientific knowledge. Solvias, one of the world’s leading independent pharmaceutical testing and manufacturing companies, understands the need for sophisticated solutions to validate the safety and efficacy of advanced new therapies, which impelled them to pursue their latest strategic acquisition to bolster their biologics and CGT capabilities. The same rationale holds for the many new alliances that have been established, such as with the National Institute of Standards and Technology (NIST) Genome Editing consortium, Bespoke Gene Therapy Consortium (BGTC) or the SMARTER project (led by Achilles Therapeutics).1,23,24,25 While Boston and California are known to be home to mature and leading pharma and biotech clusters, it is heartening to see the emergence of new geographical/regional contenders. As a case in point, Philadelphia is poised to become a top CGT cluster dubbed Cellicon Valley.26 Additionally, Catapult is the largest cell and gene therapy agglomerate in the UK and has built an impressive large-scale GMP manufacturing centre to address important manufacturing challenges.27,28 On the same side of the pond, Dr. Trudy Straetemans and her colleagues are putting in place a high-quality Cell Therapy Facility (CTF) infrastructure at the Utrecht Science Park to accelerate discoveries by bridging the gap between concept and clinical implementation. The Dutch tumor immunologist in question also initiated a new Dutch national platform (DARE-NL), which was awarded a grant of €5.3 million from the Dutch cancer fund KWF Kankerbestrijding last year.29,30 Unarguably, these commendable developments not only complement existing platforms, such as the Innovation Center for Advanced Therapies (ICAT) but also reinforces Utrecht’s role as one of the global forerunners in the field of regenerative medicine, advanced disease models and CGT.31 In the meantime, Leiden’s dynamic and vibrant Bio Science Park is attracting the likes of Bristol-Myers Squibb, one of the largest biopharmaceutical companies in the world, to build their CAR T cell therapy production center.32
In sum, with an immense potential to target and eradicate devastating diseases, cell and gene therapy developments have been advancing at an extremely rapid pace. With increased activity, and thus a burgeoning sector, it is not surprising to see increased scrutiny from regulatory authorities. As discussed throughout our blog, some of the current and most pressing challenges are related to manufacturing and quality control.2 Looking forward, it will be important for manufacturers and developers to adopt improved analytical solutions towards (1) the development of streamlined, robust GMP-compliant manufacturing processes (2) satisfying the increasingly stringent regulatory requirements and (3) consistently delivering high-quality products.
At Cergentis, we’ve made genetic QC our business. In fact, we continue to witness widespread interest in probing both [transgene] integration site and the integrity of the integrated sequence. Our Senior Director of Assay & Product Development, Harma Feitsma maintains that: “TLA-based assays prove to be excellent solutions for addressing multiple genetic QC questions at once.”.14
To learn more about our genetic QC solutions for cell and gene therapy, please visit our Products & Services page below:
1] Salcedo, R. & Rosellini, W. (2022, April 19). Hurdles ahead for cell and gene therapy makers. BioProcess International. https://bioprocessintl.com/manufacturing/cell-therapies/challenges-for-cell-and-gene-therapies-makers-face-technological-regulatory-economic-hurdles/
 Pagliarulo, N. (2021, March 16). Record funding flowed into cell, gene therapy companies last year. Biopharma Dive. https://www.biopharmadive.com/news/gene-cell-therapy-funding-2020-arm-report/596790/
 Warneck-Silvestrin, L. (2020, March 16). Gene therapies: overcoming the biggest hurdles in manufacturing. Labiotech. https://www.labiotech.eu/partner/gene-therapies-hurdles-manufacturing/
 Buente, M., Hosseini, M. & Kaltenbach, T. (2021, May 6). Leveraging the untapped potential of cell and gene therapies. Roland Berger. https://www.rolandberger.com/en/Insights/Publications/Cell-and-gene-therapies-Pharma's-next-big-wave.html
 Schneider, ME. (2022, March 16). FDA drafts guidance on genome editing, CAR T cell therapies. Regulatory Focus (A RAPS Publication). https://www.raps.org/news-and-articles/news-articles/2022/3/fda-drafts-guidance-on-genome-editing-car-t-cell-t
 U.S. Food & Drug Administration. (2022, March). Human gene therapy products incorporating human genome editing. U.S. Food & Drug Administration. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/human-gene-therapy-products-incorporating-human-genome-editing
 U.S. Food & Drug Administration. (2022, March). Considerations for the development of chimeric antigen receptor (CAR) T cell products. U.S. Food & Drug Administration. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/considerations-development-chimeric-antigen-receptor-car-t-cell-products (CAR-T)
 Balfour, H. (2022, March 25). FDA releases draft guidance for cell and gene therapy industry. European Pharmaceutical Review. https://www.europeanpharmaceuticalreview.com/news/169647/fda-releases-draft-guidance-for-cell-and-gene-therapy-industry/
 Armstrong, A. (2022, March 16). FDA's gene editing guidance is 'par for the course' but still important, analysts say. Fierce Biotech. https://www.fiercebiotech.com/biotech/fdas-gene-editing-guidance-par-course-still-important-analysts-say-crispr-intellia-verve
 Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36, 765–771 (2018). https://doi.org/10.1038/nbt.4192
 Höijer, I., Emmanouilidou, A., Östlund, R. et al. CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations. Nat Commun 13, 627 (2022). https://doi.org/10.1038/s41467-022-28244-5
 Yin, J., Lu, R., Xin, C. et al. Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing. Nat Commun 13, 1204 (2022). https://doi.org/10.1038/s41467-022-28900-w
 Raper, V. (2021, April 2). Gene therapy adopts new tools to guarantee quality. Genetic Engineering & Biotechnology News. https://www.genengnews.com/insights/gene-therapy-adopts-new-tools-to-guarantee-quality/
 de Vree, P., de Wit, E., Yilmaz, M. et al. Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat Biotechnol 32, 1019–1025 (2014). https://doi.org/10.1038/nbt.2959
 Jo, S., Das, S., Williams, A. et al. Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing. Nat Commun 13, 3453 (2022). https://doi.org/10.1038/s41467-022-30896-2
 Roth, T.L., Puig-Saus, C., Yu, R. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405–409 (2018). https://doi.org/10.1038/s41586-018-0326-5
 Merten, O.W., Hebben, M., and Bovolenta, C. (2016). Production of lentiviral vectors. Mol. Ther. Methods Clin. Dev. 3, 16017.
 Brooks, S.E., Adachi, M., Hoffman, L.M., Stein, M.R., Brooks, J., and Schneck, L. (1988). Induction of lymphomas and fibrosarcomas in nude mice after implantation of simian virus 40-transformed human meningioma. Lab. Invest. 58, 518–523.
 Reddel, R.R., Salghetti, S.E., Willey, J.C., Ohnuki, Y., Ke, Y., Gerwin, B.I., Lechner, J.F., and Harris, C.C. (1993). Development of tumorigenicity in simian virus 40-immortalized human bronchial epithelial cell lines. Cancer Res. 53, 985–991.
 Bae DH, Marino M, Iaffaldano B, Fenstermaker S, Afione S, Argaw T, McCright J, Kwilas A, Chiorini JA, Timmons AE, Reiser J. Design and Testing of Vector-Producing HEK293T Cells Bearing a Genomic Deletion of the SV40 T Antigen Coding Region. Mol Ther Methods Clin Dev. 2020 Jul 9;18:631-638. doi: 10.1016/j.omtm.2020.07.006. PMID: 32775497; PMCID: PMC7397404.
 Straetemans T, Janssen A, Jansen K, Doorn R, Aarts T, van Muyden ADD, Simonis M, Bergboer J, de Witte M, Sebestyen Z, Kuball J. TEG001 Insert Integrity from Vector Producer Cells until Medicinal Product. Mol Ther. 2020 Feb 5;28(2):561-571. doi: 10.1016/j.ymthe.2019.11.030. Epub 2019 Dec 14. PMID: 31882320; PMCID: PMC7001055.
 PharmaCompass. (2022, July 4). CDMO activity tracker: Rising demand to ensure more growth, consolidation in 2022. PharmaCompass. https://www.pharmacompass.com/radio-compass-blog/cdmo-tracker-rising-demand-to-ensure-more-growth-consolidation-in-2022?mc_cid=7d146a8a27&mc_eid=2b8e767efd
 NIST. (2022, June 9). NIST genome editing consortium. NIST. https://www.nist.gov/programs-projects/nist-genome-editing-consortium
 FNIH. Accelerating medicines partnership® Bespoke gene therapy consortium (AMP® BGTC). Foundation for the National Institutes of Health. https://fnih.org/our-programs/AMP/BGTC
 Achilles Therapeutics. (2022, July 21). Achilles Therapeutics to lead Horizon Europe-funded consortium to advance personalized cell therapy manufacturing. Achilles Therapeutics. https://ir.achillestx.com/news-releases/news-release-details/achilles-therapeutics-lead-horizon-europe-funded-consortium
 Keown, A. Why Philadelphia is poised to become a top cell and gene therapy cluster. https://biobuzz.io/why-philadelphia-is-poised-to-become-a-top-cell-and-gene-therapy-cluster/
 Catapult. Development of the largest cell and gene therapy cluster in the UK. Catapult. https://ct.catapult.org.uk/files/development-largest-cell-and-gene-therapy-cluster-uk
 Catapult. How we work. Catapult. https://ct.catapult.org.uk/Stevenage
 NWO. Utrecht CTF (Utrecht Cell Therapy Facility: National expert center ATMP development and Cellular Therapy). NWO. https://www.onderzoeksfaciliteiten.nl/node/3928
 UMC Utrecht. (2021, December 16). Over 5 million for cell and gene therapy. UMC Utrecht. https://www.umcutrecht.nl/en/over-ons/nieuws/infection-and-immunity/dec-16-over-5-million-for-cell-and-gene-therapy
 Utrecht University. ICAT (Innovation Center for Advanced Therapies). https://www.uu.nl/en/icat
 Bristol Myers Squibb. (2021, October 28). Bristol Myers Squibb starts construction of its European therapy facility, at Leiden Bio Science Park. Bristol Myers Squibb. https://www.bms.com/nl/en/news/news-releases/car-t-production-centre-leiden.html