Refining the molecular blueprint of in vivo CAR-T cell therapies
Following the first commercial approvals in 2017, the chimeric antigen receptor (CAR) T-cell therapy market has expanded rapidly thanks to the significant impact these treatments have had on patient outcomes in hematologic cancers, and is projected to keep growing as CAR T-cell therapies are administered earlier in the patient’s treatment journey, and target indications expand from oncology into autoimmune conditions.
Reprogramming a patient’s T-cells in vivo is a promising approach to transforming the accessibility of these life-saving therapies to a larger patient population, offering several advantages:
· patients can be treated in a single hospital visit
· treatments will be available “off-the-shelf”
· manufacturing processes are less complicated and more cost effective
Manipulating Tropism for Precision Targeting
The most commonly pseudotyped lentiviral envelope protein, Vesicular Stomatitis Virus Glycoprotein (VSV-G)targets the low-density lipoprotein receptor (LDLR), which is found on thesurface of most mammalian cells. This is a problem for in vivo therapies, where precise tissue targeting is critical for safety and efficacy; indeed it is essential that the viral vector reserves its cancer-fighting payload forthe T-cells it’s intended to reprogram.
To re-engineer tropism, lentiviral vectors can be “dressed” in a new envelope protein that has a different mechanism of viral entry and naturally targets a different cell type (2). However, while there are viruses (eg HIV) that can transduce T cells, thereisn’t a known virus with a specific enough natural tropism that it can be used for in vivo CAR T-cell modification without further engineering to blind the envelope protein to its natural receptor, and re-target it towards T-cells (3).
Engineering Biological Safety Mechanisms
Once the lentiviral envelope is engineered to specifically target T cells for transduction, the question becomes whether this is enough to reduce the risk of off-target CAR expressionin an in vivo therapy. So far, the general scientific consensus seems to be “why risk finding out?”.
The addition of tissue specific promoters to the CAR construct represents a “belt and braces” approach to biological safety. As well as adding an extra layer of biological safety, ongoing research is exploring whether the addition of tissue specific promoters can also enhance efficacy.
CAR T-cell treatment causes serious side effects including cytokine release syndrome, tumour lysis syndrome and on-target off-tumour toxicity. While it may not be possible to avoid these side-effects, it is possible to engineer a molecular “emergency brake” into the CAR construct, so that should side effects become intolerable or life-threatening, or unexpected toxicities occur, treatment can be stopped (4,5). This may be especially important for in vivo CAR T-cell therapies, where the risk of uncontrolled T-cell production is somewhat higher than for ex vivo approaches.
The risk of insertional mutagenesis from lentiviral vectors is typically low, and the use of self-inactivatingviral backbones reduces this further, but the risk-reward ratio changes when CAR T-cell therapies stop being considered an “end of line” treatment or are used to treat non-fatal conditions. At this point any risk of insertional mutagenesis is unacceptable.
Non-integrating lentiviruses may offer a solution. However, while the risk of insertional mutagenesis is eliminated, so is the potential to maintain expression of the transgene throughcell division. The “holy grail” of safe lentiviral vector design is a non-integrating lentivirus whose transgene expression can still be maintained throughout multiple cell divisions, as the activated CAR-T cells divide as they fight the cancer. Researchers at ViroCell Holdings are currently working on the identification of novel regulatory elements and sequence motifs to achieve this persistence with non-integrating lentiviruses, an endeavour in which they wouldwelcome additional scientific collaboration (6).
Purity and Potency: The Backbone of Manufacturability
Residual host cell proteins or DNA can be “washed off” reprogrammed T-cells prior to re-infusion with an ex vivo therapy, but there’s no such opportunity for clean-up of the vector prior to injection of an in vivo therapeutic. This means the manufacturing process must be optimised for production of mature, functional lentiviral vectors and to minimise production of non-functional byproducts.
The starting point for vector purity once again begins at the molecular level. Assembly of mature lentiviralparticles requires the Gag protein to recognise and interact with a specific sequence on the RNA genome, carrying it to the cell membrane for budding. This process is not always executed perfectly; lentiviral particles may form that are only partially packaged, completely empty or contain extraneous cellular RNA. Simultaneously, additional extracellular vesicles (EV) are also likely to bud from the cell, with or without viral RNA inside, and most likely with viral envelope proteins on the EV surface(7).
Some of this incorrect packaging is caused by aberrant splicing of the lentiviral genome. This can result in unwanted “breakthrough” expression of the CAR, which can end up on the surface of the manufacturing cell, and from there also contaminating the lentivirus with CAR, in a phenomenon known as aberrant pseudotyping. This has the potential to negatively affect vector potency and safety, especially in vivo. Lentitek is tackling this problem using a proprietary promoter that sits upstream of the 5’ LTR and produces a viral genome that has been demonstrated to be resistant to splicing, and therefore to unwanted expression of the CAR payload during lentivirus production(8).
How Plasmid Design Software and Collaborative Project Management Enable Complex Workflows
There is no question that designing a lentiviral vector encoding a CAR construct for in vivo use is a major feat of genetic engineering. It’s also clear that there is going to be no single “right” way to go about it; every approach will have to balance improvements in precision or safety, with potential decreases in functional titer or other complications. Research teams involved in this endeavour are going to need to collaborate closely, and work through multiple iterative rounds of trial and error to identify the optimal vector design. Plasmids are likely to be sourced and constructed from institutions around the world, making careful annotation necessary to keep track of provenance, usage and outcomes. ViroCell Holdings approach this using a combination of intelligent design, computational biology tools and in vitro evolution, combined with selection strategies, to develop lentiviral vectors that are specifically optimized for their planned application.
This is where digital lab management technology like Lab Thread can help by providing a robust digital infrastructure for scientific discovery, project management and collaboration. This is also an area where new IP is constantly being generated, and here again, digital records and timestamped records of relevant discoveries can be fundamental in proving novelty and priority. Advanced plasmid design software and other molecular biology tools support complex and collaborative genetic engineering with “in sequence” commenting functionality for detailed annotation, and sequence sharing tools to streamline collaboration.
Using the software to link each design to its experimental outcome and sample location helps manage the assessment of each genetic variable involved in complex plasmid engineering, streamlining the process of iterative design and testing to optimise the construct. Meanwhile, connecting molecular design software to a complete digital record of where each plasmid design has been used—which experiments, cell lines, bioreactor runs etc—makes it infinitely easier to assemble a fully traceable regulatory package or patent specification when the time comes. Comment, feedback and collaboration tools at the sequence, design, task and project level facilitate ready communication within and between teams, while a “big picture” vision of project progress ensures that collaborative project management is supported throughout the entire program.
Plasmid design software can’t build a perfectly optimised construct for an in vivo CAR T-cell therapy, but it can make the lives of the scientists doing so considerably easier through detailed molecular design, collaborative project management, easily accessible data, and comprehensive sample tracking.
Could state-of-the-art molecular biology tools and collaborative project management enhance your science? Sign up to Lab Thread now!
References
1. https://www.marketsandmarkets.com/Market-Reports/car-t-cell-therapy-market-47772841.html
2. Duvergé A, Negroni M. Pseudotyping Lentiviral Vectors: When the Clothes Make the Virus. Viruses. 2020 Nov 16;12(11):1311. doi: 10.3390/v12111311. PMID:33207797; PMCID: PMC7697029.
3. Mhaidly R, Verhoeyen E. The Future: In Vivo CAR T Cell Gene Therapy. Mol Ther. 2019 Apr 10;27(4):707-709. doi: 10.1016/j.ymthe.2019.03.012.Epub 2019 Mar 23. PMID: 30914238; PMCID: PMC6453545.
4. Moghanloo E, Mollanoori H,Talebi M, Pashangzadeh S, Faraji F, Hadjilooei F, Mahmoodzadeh H. Remote controlling of CAR-T cells and toxicity management: Molecularswitches and next generation CARs.Transl Oncol. 2021 Jun;14(6):101070. doi: 10.1016/j.tranon.2021.101070. Epub2021 Mar 28. PMID: 33789222; PMCID: PMC8027274.
5. Głowacki P, Rieske P. Application and Design of Switches Used in CAR. Cells. 2022 Jun 13;11(12):1910. doi: 10.3390/cells11121910. PMID:35741039; PMCID: PMC9221702.
6. Farzaneh, F, Ostrout N. De-Risking Cell and Gene Therapies with Innovative Solutions: A Reviewfor Leveraging a Proven Workhorse Technology in New Ways. Pharma’s Almanac. 2025. Jul;35
7. Barbieri E, Heldt CL. Challenges and Opportunities in Lentivirus Viral Vector Manufacturingfor In Vivo Applications. Biomedicines.2026 Feb 5;14(2):369. doi: 10.3390/biomedicines14020369. PMID: 41751268; PMCID:PMC12938532.
