*Guest blog by Lab Thread. If you would like to submit a guest blog please email [email protected].
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-inactivating viral 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 through cell 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 would welcome additional scientific collaboration (6).
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