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Maximizing Efficiency

Date: November 2024

Getting a transgene to be expressed inside the target cell is the name of the game.


The leading delivery vehicle for in vivo gene therapy, recombinant adeno-associated viruses (rAAVs) are transforming the health of people with genetic diseases. And as therapies using rAAV vectors reach an ever-growing number of patients with more complex diseases, it becomes increasingly important to maximize the efficiency of AAV-based gene therapies.  

This can be achieved by improving a number of factors, including tissue specificity, the transduction of the target cell, and the packaging and expression of the transgene.

Because the capsid – the protein shell of rAAV vectors – is a key component in determining tissue specificity as well as facilitating entry of rAAV vectors into a target cell, a number of key ways to improve the efficiency of AAV-based gene therapies center on the capsid.

Natural Discovery

One way to improve the therapeutic effects of gene therapy using rAAV vectors is to discover new natural AAV variants and screen for those with capsids that have optimal transduction and tissue specificity while, at the same time, are able to escape pre-existing antiviral antibodies.

Finding a naturally occurring AAV variant that fulfills all of these criteria is tricky, as many AAVs from non-human samples tend to have limited tissue tropism, whereas AAVs from human samples are more likely to be neutralized by pre-existing antiviral antibodies.

Directed Evolution 

Another approach for maximizing transduction efficiency is directed evolution. This is a technique by which pressure is imposed on a known AAV variant with the intention of creating a newly derived variant with a capsid that exhibits a specific, selected-for improvement, such as increased transduction or tropism for a particular tissue or cell type.

One form of directed evolution is DNA shuffling, an approach by which the capsid genomes from different AAV serotypes are fragmented and reassembled. Although this has the advantage of generating beneficial mutations quickly, one drawback of directed evolution, in general, is that many methods are not cross-species translatable

Rational Design

In addition, it is possible to improve transduction efficiency of an rAAV vector by modifying its capsid, a strategy known as rational design. This approach includes point mutations of specific amino acid residues, chemical modifications that do not change the amino acid composition, and the insertion of non-viral domains into the capsid. These changes can lead to enhanced gene delivery to the target organ or tissue as well as the detargeting of specific tissues, although modifications do sometimes lead to decreased transduction.

Beyond the Capsid

Computational tools can be used to modify not only the capsid of an rAAV vector but the genome itself. For example, algorithms can be used to identify codons that, if replaced, would enhance the translation of the transgene and generation of the functional protein. Another way to improve tissue-specificity is the use of a cell type-specific promotor thus targeting the transgene expression to the corresponding cell type or tissue.

There are also decisions about the production process to consider in order to maximize transduction efficiency while optimizing other crucial factors such as safety, cost, scalability, and flexibility. Currently, the leading method for manufacturing rAAVs relies on transient transfection, an approach that, while flexible and versatile, is costly and lacks scalability.

Alternative approaches entail infecting either insect or mammalian cells with the virus. One of these approaches, which relies on stable cell lines, carries a low cost and is scalable, however, it is also complex to produce and must be serotype- and vector genome-specific.

A key challenge for next-generation manufacturing platforms is to reduce the number of empty or partial rAAV capsids that are produced while still increasing the overall production yield. Because empty and partial capsids do not confer any therapeutic benefit to a patient but may trigger an immune response, reducing them is essential for increasing the vector potency while minimizing the potential for immunotoxicity.

Safety Above All

From optimizing the design of rAAV vectors to the process by which they are manufactured, there are many promising ways to increase the efficiency of AAV gene therapy. Since safety is paramount, it is important to demonstrate the efficiency of a new strategy in vitro using cell-based assays in accordance with FDA guidelines before transferring it to a clinical trial.

Reference

Wang, JH., Gessler, D.J., Zhan, W. et al. Adeno-associated virus as a delivery vector for gene therapy of human diseases. Sig Transduct Target Ther 9, 78 (2024).

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