Visionary Science: Gene therapy saves sight thanks to animal research

Yesterday the BBC News and Guardian Newspaper reported that a team led by surgeon Professor Robert Maclaren at the Oxford Eye Hospital had succeeded in using gene therapy to halt the decline in vision in six patients with the progressive eye disorder choroideremia.

All six patients were taking part in a clinical trial, and what was especially exciting was the sustained improvement in vision in the two patients whose vision had deteriorated the most. This is great news for the patients themselves, and as the technique is likely to be applicable to many different genetic eye disorders it is also good news for many millions of people who may benefit in future. It is also an excellent example of how years of research in mice, dogs and monkeys can lead to an important clinical advance.

Choroideremia is caused by a defect in the CHM gene, which encodes the Rab escort protein 1 (REP1), and lack of this protein leads to gradual degeneration of the retinal epithelium layer  (RPE) and rod photoreceptor cells in the eye, causing a progressive decline in vision that usually starts with night blindness and loss of peripheral vision, and eventually leads to total blindness.

To halt this decline Professor Maclean’s team used a vector  based on a modified adeno-associated virus serotype 2 (AAV2) which could express the healthy CHM gene in the eye and produce REP1.  Why did they choose AAV2 out of all the potential virus vectors available? The Lancet paper reporting on this trial cites a key study published in the Journal of Molecular Medicine in  2013* by Professor Maclaren and colleagues, which describes the development and evaluation of the vector used in the trial. In their introduction and discussion they discuss the rational for choosing the AAV2 vector:

With a functional fovea, safety with regard to avoiding a vector-related inflammatory reaction is of paramount importance. Two recent clinical trials had demonstrated that serotype 2 adeno-associated viral (AAV2) vectors have no long-term retinal toxicity when administered at the dose range 1010–1011 genome particles [12, 13]. Importantly, in addition to transducing the RPE, AAV2 is also known to target rod photoreceptors efficiently in the non-human primate [14], providing the ideal tropism for a CHM gene therapy strategy.

… Although one might argue that other serotypes such as AAV8 may be more efficient in targeting photoreceptors, AAV2 with the CBA promoter remains the gold standard for retinal transduction as evidenced by the sustained vision in Briard dogs treated with AAV2 vector over a decade ago [35].

12. Cideciyan AV, Aleman TS, Boye SL, Schwartz SB, Kaushal S, Roman AJ, Pang JJ, Sumaroka A, Windsor EA, Wilson JM, et al. Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proc Natl Acad Sci U S A. 2008;105:15112–15117. doi: 10.1073/pnas.0807027105.  13. Jacobson SG, Cideciyan AV, Ratnakaram R, Heon E, Schwartz SB, Roman AJ, Peden MC, Aleman TS, Boye SL, Sumaroka A, et al. Gene therapy for Leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years. Arch Ophthalmol. 2011;130:9–24. doi: 10.1001/archophthalmol.2011.298.  35. Bennicelli J, Wright JF, Komaromy A, Jacobs JB, Hauck B, Zelenaia O, Mingozzi F, Hui D, Chung D, Rex TS, et al. Reversal of blindness in animal models of Leber congenital amaurosis using optimized AAV2-mediated gene transfer. Mol Ther. 2008;16:458–465. doi: 10.1038/

So which two clinical trials are they referring to? Well, as you can see from the references they are referring to the successful trials of gene therapy for Leber Congenital Amaurosis (LCA)whose development we discussed on this blog back in 2009. As McLaren and colleagues point out, the sustained expression of RPE65 and long-term recovery of vision in the Briard dog model of LCA was a key factor in their decision.  The observation that AAV2 could be used to drive gene expression in rod photoreceptors was also important, as Maclaren and colleagues had previously generated a genetically modified mouse model of Choroideremia by knocking out CHM expression in the eye, and established that in Choroideremia the degeneration of rod photoreceptors is independent of the degeneration of the RPE, so it is crucial that the vector can drive healthy gene expressed in both the rods and RPE.

To develop the vector Maclaren and colleagues first compared the efficiency of 3 different promoters (promoters are sections of DNA that promote gene expression) -AAV2/2-EFS, AAV2/5-EFS and AAV2/2-CBA  – in driving expression of the CHM gene when added in vitro in a variety of dog and human fibroblast (connective tissue cell)  lines in an AAV2 vector, and then when injected in vivo in the retinas of healthy mice. These studies demonstrated that the most efficient AAV2 vector – named AAV2/2-CBA-REP1 – could drive expression of high levels of REP1 in both the RPE and rod photoreceptors of mice. After identifying the most effective AAV2 vector for expressing REP1  they assessed whether it was capable of expressing REP1 in isolated human retina’s obtained post-mortem from human donors, which it did. They then evaluated whether there as any toxicity associated with expressing REP1 in vivo in the retina of healthy mice, finding that AAV2/2-CBA-REP1 was non-toxic even when injected into the retina at high doses, and that it did not adversely affect vision.

Following these studies the question remained; would injection of AAV2/2-CBA-REP1 stop deterioration of vision in choroideremia?

To address this Maclaren and colleagues turned again to the genetically modified mouse model of choroideremia thay they had created earlier. Injection of the vector into the retinas of these CHM mice:

Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham injected eyes confirming that AAV2/2-CBA-REP1 is a promising  vector suitable for choroideremia gene therapy in human clinical trials.”

In other words the therapy worked in the mouse model of choroideremia, paving the way for the successful clinical trial reported this week.

This new therapy is another example of the importance of animal studies to the development of new clinical techniques and therapies, but also highlights the fact that medical science is a long game, with basic and applied research conducted more than a decade, even two decades,  ago being crucial to this week’s exciting announcement. This is something policy makers would do well to remember!

Paul Browne

* While this paper was published in 2013, the work it reports was completed several years earlier, before the clinical trial was launched in 2011.

1) Tanya Tolmachova, Oleg E. Tolmachov, Alun R. Barnard, Samantha R. de Silva, Daniel M. Lipinski, Nathan J. Walker, Robert E. MacLaren,corresponding author and Miguel C. Seabra “Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo”  J Mol Med (Berl). 2013 July; 91(7): 825–837. PMCID: PMC3695676

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