Another excellent example of gain of function research is modification of a viral vector to make it more useful for human gene therapy.
Adenovirus associated virus (AAV) is the most commonly used vector for a variety of gene therapy applications, including gene replacement and gene editing. These small viruses, which comprise a single-stranded DNA genome surrounded by an icosahedral protein shell (illustrated), have a number of features that make them useful for gene therapy, including easy manipulation and growth in large quantities, and induction of persistent gene expression for long periods of time. An example is the drug Luxturna which is an AAV vector containing a retinal pigment gene that is used to treat some forms of blindness.
Naturally occurring AAV vectors have some limitations, including the propensity to become sequestered in the liver after systemic injection. Consequently reaching other organs with AAV vectors requires injection of large amounts of recombinant virus, which may be accompanied by toxicity.
A number of approaches have therefore been developed to modify AAV vectors so they preferentially infect other tissues. A number of muscle diseases would benefit from gene therapy and therefore AAV vectors have been developed to target that tissue. In one approach that I described before, ancestral AAV viruses were recovered and shown to efficiently infect muscle cell types.
A more recent approach involved modifying the AAV genome by inserting random stretches of 7 amino acids into the viral capsid protein, infecting mice with large libraries of these variants, and identifying those that best infect muscle. In addition, muscle-specific promoter sequences were also incorporated in the viral genome. After multiple rounds of infection of mice, recovery of viruses from muscle and reinfection, capsids were identified that more efficiently infect muscle tissue and less efficiently infect liver, in multiple mouse strains and in nonhuman primates. The new AAV capsids can rescue two different types of muscle disease in mice: Duchenne muscular dystrophy and X-linked myotubular myopathy after intramuscular inoculation.
The ability of these new AAV vectors to preferentially infect muscle cells depends on the presence of three added amino acids in the viral capsid, RGD. This three amino acid motif is known to bind cell surface proteins called integrins. Indeed, the increased efficiency of these engineered vectors depends on the presence of integrins on target cells.
These findings not only have produced better AAV vectors for targeting muscle tissue, but establish a strategy to engineer viruses to preferentially infect any tissue. The new vectors are the product of gain of function research: the original AAV has been given new properties. This work constitutes another example of the broad potential benefits of gain of function research in virology.
Adenovirus associated virus (AAV) vectors are being increasingly used for gene therapy because they are not pathogenic in humans and persist for long periods in certain cell types. Currently 120 gene delivery clinical trials with these vectors are in progress, and two have been approved: Luxturna to treat a rare form of blindness, and another for the treatment of spinal muscular atrophy. Despite these successes, improvements of the efficiency of gene delivery by these vectors are needed. In silico reconstructions of putative ancestors of AAV has led to the 