Non-viral vectors precision tuned for RNA therapies
Optimer can act as non-viral vectors and precisely target specific tissue and cell types to deliver conjugated RNA therapies right into the cell.
As RNA therapies continue to increase in clinic and see more and more approvals, this modality is proving that we now can start to address disease-causing mutations and protein imbalances.
RNA-based therapies modify disease pathways in the body, by silencing, activating or changing the specific RNA transcripts, editing their message to provide new messages that help to overcome the disease causing code.
RNA-based therapies can potentially target any gene of interest as long as the exact sequence on the target RNA to be modified is known and there is a method to deliver the RNA therapy to the right cell type in the body to effect the target transcripts. This breaks the problem down into two specific issues, tailoring the sequence and activity of the RNA therapy itself and delivering it to its site of action in the body. Now RNA therapies are seen to be highly effective once at their target site, but delivering them into the right target cells to have this effect, remains the major challenge limiting this modality.
The delivery challenge
Targeted delivery remains a major translational challenge for RNA therapies, with three major hurdles:
- Targeting to the specific cell type
- Allowing the RNA therapy to penetrate the target cell
- Ensuring the therapy is functional once in the cell
Targeting to the specific tissue and cell type is important to increase the amount of drug at the site of action for increased effect and to reduce off-target interactions that give rise to unwanted side effects.
Once the RNA therapy is at the right location in the body, it’s important to be able to get it across the cell membrane to have its effect. With RNA therapies being highly negatively charged and larger than traditional small molecule drugs they don’t naturally cross the cell membrane. Receptor-mediated internalisation allows internalisation according to the specific biomarkers expressed on target cells.
The final challenge for RNA therapies is to escape the endosome. Vesicles budding from the cell membrane can fuse with the early endosome, which sends components for lysosomal degradation, thus degrading the RNA therapy before its had the chance to interact with cellular machinery to have its effect. So, the goal at this stage is to allow the RNA therapy to escape from the endosome and exert its function in the cytoplasm.
Optimer development for use as non-viral vectors includes tailoring of the molecule to help overcome each of these hurdles.
Tuning Optimer delivery vehicles
The discovery and development process for Optimer non-viral vectors allows tailoring for:
- precision targeting
- affinity
- cell internalisation
- functionality in blood
- half-life
Following this all Optimers can be conveniently conjugated to RNA therapies via a variety to allow targeting.
Target or phenotype directed non-viral vectors
For discovery of Optimer to act as non-viral vectors we can take two approaches.
If a specific receptor target on the cell surface is known, we can develop Optimer binders to that receptor through protein selection, conjugating the target to beads and processing this through multiple rounds of screening, including the target of interest and counter-targets to remove cross-reactivity.
This discovery process is performed in a relevant matrix, such as plasma, to remove cross-reactive binders to plasma components. Once an Optimer is discovered to the specific receptor we carry out further selection rounds to whole cells that express the protein. This ensures the Optimer non-viral vector binds to the receptor in its native, cell-expressed form.
If a receptor is not known or you would like to explore the potential of novel biomarkers to target for delivery, it is possible to perform vector discovery in a phenotype-directed way with Optimer. Screening against, for example healthy vs disease, or different cell types, we are able to isolate Optimer vectors that are specific to the required cell type. The specific receptor to which they bind can then be identified by pulldown and mass spectrometry following discovery.
Ensuring internalisation
Internalisation into the target cell type is built into the Optimer discovery process. During discovery Optimer non-viral vectors are discovered for the ability to selectively bind the target receptor as a protein and on the cell surface, along with their internalisation to target cells. Any Optimer vectors that don’t internalise to the target cell or that internalise to non-target cells are removed from the discovery process.
This internalisation into target cell types can be optimised for cell-specific internalisation without internalising into off-target cell types, specific timeframes of internalisation to allow rapid uptake or slower uptake, and also for the internalisation of the entire Optimer-RNA therapy conjugate, to make sure the therapeutic is internalised along with the Optimer non-viral vector unit.
Vector stability and half-life
Optimer non-viral vectors can be tuned for in vivo stability and half-life to meet different therapeutic strategies.
Stability can be tuned to allow degradation once the therapy has had sufficient time to reach its site of action and achieve its therapeutic effect. Incorporation of 2’-fluoro or 2’ O-Methyl groups into the Optimer backbone reduce endonuclease degradation, while capping of termini or circularisation of the Optimer vector reduce exonuclease degradation. These modifications can be scaled to optimise stability.
To alter circulating half-life Optimer non-viral vectors can be fitted with a half-life extension module, like an albumin-binding domain, that can extend half-life for longer residency times. On the other hand, for rapid hit-and-run strategies the small size of the Optimer alone allows for renal filtration, removing the Optimer-RNA therapy from the body, which can limit off-target effects.
Balancing affinity for tissue penetration
Affinity requirements for Optimer non-viral vectors depend on the target receptor expression in specific tissues.
- Highly expressed receptors: Intermediate affinity allows improved tissue penetration
- Low expression of receptor: High affinity required to locate receptor in tissue
Affinity is tuned during the development process with Optimers with the desired affinity profiles selected and those with too high or too low affinity discarded at this stage. Our standard development process also includes truncation of the full length Optimer to its smallest fragment, and the increased stabilisation and removal of unnecessary sequence from the molecule can help to increase stability if required.
The small size of Optimer vectors (10th-15th of standard antibody) also supports improved tissue penetration.
Escape from the endosome
Optimer delivery vehicles are compatible with both cleavable and non-cleavable linkers, and can be conjugated using diverse site-directed chemistries. Using different linkers in Optimer-conjugates enables different strategies for therapeutic release and testing of multiple linkers can easily be incorporated into Optimer development processes.
Activity and interest in RNA therapies continues to grow, despite the challenge of targeted delivery. Optimer non-viral vectors can be tuned according to the disease and drug requirements to deliver increased on-target and reduced off-target effects for this modality to deliver its optimal therapeutic potential.
For more information watch the on-demand webinar