With more than 5,000 gene therapies in the clinic today [link], decades of promise finally seem to deliver treatments for dozens of rare diseases. Safety and effectiveness are however still major concerns, with short-lived and off-target expression being the main roadblocks [link]. And no matter how well-designed a gene therapy is, if it doesn’t get switched on in the right place at the right time, it is all in vain. To improve these factors, manufacturers can adjust three principal components: the therapeutic gene, the vector and the promoter.
When discussing specificity, Annogen’s customers often name vectors as their principal tailoring strategy. In this regard, viral vectors including adeno-associated virus (AAV) and lentivirus are most commonly used for gene therapy delivery. While there are differences in tissue-specificity, any viral vector raises concerns on potential off-target effects, i.e. when tissues other than the target tissue are effected upon administration. As such, there is only so much control on tissue specificity that can be performed with vector selection.
The cargo delivered by viral vectors not only contains the therapeutic gene, but essentially also a promoter and in some cases an enhancer. Promoters are regulatory elements that ensure the therapeutic gene is correctly expressed in the targeted tissue. To date [link and link], the majority of all clinical trials use one of the ubiquitous viral promoters including CMV (cytomegalovirus), CBA (chicken beta-actin) or CAG (a synthetic promoter consisting of CMV enhancer, CBA promoter and a rabbit beta-globin splice acceptor). This characteristically drive high expression, which can be beneficial in certain cases, though off-target transduction is a side-effect and one of the leading causes for toxicity [link]. This indicates the field is still conservative in choosing regulatory elements and prefers to stick with flavours known to be accepted by the regulatory authorities.
In contrast, constitutive human promoters such as elongation factor-1 alpha (EF‐1α) tend to have lower overall activity while ubiquitously expressed, thereby minimizing off-target effects and thus improving safety. Simultaneously, cell health may be compromised when a transgene is overexpressed, further motivating the use for dosage-controlled or tissue-specific promoters. EF‐1α is especially useful in conditions where other promoters (including CMV) have diminished activity or have been silenced, such as in hepatocytes (link).
Promoters can be chosen to be active only in specific cell types to avoid expression in other tissues, to maximize safety in case delivery to health tissue occurs. Indeed, up to 2022, of the 27 unique promoters used in clinical trials were disclosed [link], the vast majority were endogenous and tissue-specific. Different therapeutic areas are notably exploring tissue-specific promoters at different speeds, with disorders in the CNS falling behind considerably. From interactions with biopharmaceutical developers, we however know that there is a need for such elements for use in CNS-specific advanced therapies. We furthermore learned over time that, more generally, many biopharmaceutical often dedicate considerable efforts to identify such elements in-house, though these experiments have been considered inefficient and non-exhaustive.
While not typically a concern with gene therapy, tissue-specificity may furthermore help avoiding leakage in case of using conditional promoters, which we will touch upon in a future blog concerning cell therapy development.
When choosing a promoter for a gene therapy, another consideration is silencing of the delivered gene. This is a widely known phenomenon [link] that can drastically reduce the therapeutic effect [link]. Methylation of the exogenous promoter (termed “promoter attenuation”) is the main cause of silencing, as was experienced with early gene therapies for cystic fibrosis [link]. Short-lived, transient expression of the transgene would necessitate multiple treatments, yet that would in turn likely lead to toxicity, inflammation and increased cost of treatment. This again raises concerns on the use of ubiquitous promoters, as the CMV enhancer used in CAG and CMV promoters, has shown to cause silencing of the viral transgene [link and link]. Therefore, utilizing promoters with lower susceptibility to methylation are key and motivate cell therapy developers to move away from using such generic regulatory elements.
In conclusion, effectiveness and safety (= success) of a gene therapy are dependent on choosing the right vector and promoter. Considerations in choosing a regulatory element are dosage level, tissue-specificity and avoiding promoters that can be silenced by methylation should be a priority. Bespoke promoters tailored for specific indications are considered the preferred method for ensuring success of future gene therapies.
Mastering the discovery of the right promoters and enhancers can be the difference between churning out industrial quantities of product or ending up with a science-fair experiment gone wrong. At Annogen, we have made regulatory elements our life’s work by experimentally identify and validate the best promoters, exactly tailored for our customers. Our customers get first-pick on exclusively licensing these elements, thereby ensuring effectiveness of their therapy and obtaining a competitive edge over their peers.
Contact Annogen today for your high-performance promoters.
