Directed evolution of novel adeno-associated viral vectors for gene therapy

Published on August 5, 2014   52 min

Other Talks in the Series: Gene Transfer and Gene Therapy

0:00
This is David Schaffer. I'm a professor at the University of California at Berkeley in Chemical and Biomolecular Engineering, Bioengineering, and the Helen Wills Neuroscience Institute, as well as director of the Berkeley Stem Cell Center. In addition, I'm co-founder of the company, 4D Molecular Therapeutics. And I'm going to be talking about directed evolution of novel adeno-associated viral vectors for gene therapy.
0:23
So if we assess the current status of the field of gene therapy, which can be defined as the introduction or delivery of genetic material to the cells of an individual for therapeutic benefit. There have been a number-- an increasing number of successes over the past few years, and these include bona fide clinical successes for the treatment of Leber's congenital amaurosis, hemophilia B, as well as familial lipoprotein lipase deficiency and x-linked adrenoleukodystrophy. Now the first three indications I mentioned were made possible through adeno-associated viral vector technology, which has been increasingly successful as we mentioned over the past few years. Despite all of these successes however, a number of disease targets are still beyond the reach of current gene transfer technology. And that technology must be made better, which is the focus of my talk today.
1:09
So if we zoom into more detail next slide on adeno-associated virus, we see that this virus has a relatively simple genome. It simply contains two Cis elements-- the inverted terminal repeats that flank the genome. And the genome only has two genes that are open reading frames-- REP, which encodes several proteins, as well as CAP, which encodes three proteins, as well as a fourth protein and an alternative reading frame. Now this virus encodes as I mentioned several versions or several variants of the cap protein-- 60 copies of which will self assemble to create this beautiful icosahedral structure-- and the rep protein which is responsible for mediating the replication as well as of other functions of the viral genome will then load this capsid with it's viral genome payload for cargo. So this virus is non-pathogenic. It's never been associated with human disease. It's among the smallest of viruses both in terms of genome size at around 4.9 kilobases of information, as well as the dimensions of the particle-- around 25 nanometers in size. And a number of natural variants of the virus have been isolated, which typically have variations in the amino-acid sequence or identity on these exposed loop regions that you can see on the viral capsid structure.
2:21
Now the way that this was converted from a virus into a gene delivery vehicle in the late 1980s was by simply cutting the viral genes added the viral genome, and pasting and replacing it onto a separate piece of DNA. And furthermore, because as its name implies, AAV requires the presence of an adenovirus in order to replicate. We also need some adenoviral helper functions, or helper genes. And these three constructs, which are delivered often as plasmids, are introduced into a producer cell line. The REP and CAP mediate viral replication and formation of the capsis, which are then loaded with the gene of interest as the cargo. The resulting gene delivery vehicles or vectors have been shown to be extremely safe. There's a starting point that's greater than 90% percent of the human population has been naturally exposed to at least one serotype of AAV. And again, it's not associated with human disease. And as a result, the vectors based upon AAV have a positive safety profile, and there had not been vector adverse events, or serious adverse events within clinical trials associated with AAV. In addition, it's been shown to be highly efficient in mediating gene delivery in a number of trials to muscle, liver, lung, brain, and retina. After delivering its genome to the nucleus of a non-dividing cell results in very stable expression of a protein or RNA product. And it's showing efficacy in increasing number of clinical trials.
3:40
So in the next slide, we can see several publications associated with these trials. So work from leading labs within the field have shown in, for example, two New England Journal of Medicine articles. Efficacy for the treatments, or in gene therapy, to treat rare blinding disease, Leger's Congenital Amaurosis, Type 2. And furthermore, efficacy for AAV mediated gene therapy to treat hemophilia B. Furthermore, these clinical successes have somewhat culminated with the relatively recent market approval, clinical approval, within the EU-- the European Union-- of a gene therapy to treat like a protein life-based efficiency that was developed by Unicure, a company based in Amsterdam. So as an example of these successes, as we move on to the next slide.
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Directed evolution of novel adeno-associated viral vectors for gene therapy

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