Gene therapy for red–green colour blindness in adult primates

Katherine Mancuso1, William W. Hauswirth2, Qiuhong Li2, Thomas B. Connor3, James A. Kuchenbecker1,Matthew C. Mauck3, Jay Neitz1 & Maureen Neitz1

1Department of Ophthalmology, Box 356485, University of Washington, 1959 North East Pacific Street, Seattle, Washington 98195, USA. 2Department of Ophthalmology and Powell Gene Therapy Center, University of Florida, 1600 South West Archer Road, Gainesville, Florida 32610, USA. 3Department of Ophthalmology, Medical College of Wisconsin, 925 North 87th Street, Milwaukee, Wisconsin 53226, USA.

Red–green colour blindness, which results from the absence of either the long- (L) or the middle- (M) wavelength-sensitive visual photopigments, is the most common single locus genetic disorder.
Here we explore the possibility of curing colour blindness using gene therapy in experiments on adult monkeys that had been colour blind since birth. A third type of cone pigment was added to dichromatic retinas, providing the receptoral basis for trichromatic colour vision. This opened a new avenue to explore the requirements for establishing the neural circuits for a new dimension of colour sensation. Classic visual deprivation experiments
1 have led to the expectation that neural connections established during development would not appropriately process an input that was not present from birth. Therefore, it was believed that the treatment of congenital vision disorders would be ineffective unless administered to the very young. However, here we show that the addition of a third opsin in adult red–green colourdeficient primates was sufficient to produce trichromatic colour vision behaviour. Thus, trichromacy can arise from a single addition of a third cone class and it does not require an early developmental process. This provides a positive outlook for the potential of gene therapy to cure adult vision disorders.

Gene therapy was performed on adult squirrel monkeys (Saimiri sciureus) that were missing the L-opsin gene. In this species, some females have trichromatic colour vision whereas males are red–green colour blind2. Serotype 2/5 recombinant adeno-associated virus (rAAV) containing a human L-opsin gene under the control of the L/M-opsin enhancer and promoter (Fig. 1a) was delivered to the photoreceptor layer by subretinal injections (see Methods).

Transcriptional regulatory elements were chosen to direct expression preferentially in M cones, but not short- (S) wavelength-sensitive cones or rods3. To provide the receptoral basis for trichromacy, animals received three 100-ml injections (containing a total of 2.731013 viral particles) in each eye, which produced a relatively uniform, third submosaic of approximately 15–36% of M cones that coexpressed the transgene.

Before treatment, monkeys were trained to perform a computerbased colour vision test, the Cambridge Colour Test4,5, which was modified for use with animals6. Dichromats who are missing either the L- or the M-photopigment fail to distinguish from grey colours near the so-called 'spectral neutral point' located in the bluegreen region of colour space (near dominant wavelength of 490 nm) and complementary colours near the 'extra-spectral neutral point' in the red-violet region (near dominant wavelength of2499 nm).

Whereas trichromats have the four main hue percepts blue, yellow, red and green, dichromats only have two percepts, nominally blue and yellow. Before treatment, two dichromatic monkeys completed three colour vision tests consisting of 16 hues (Fig. 2b, c). Four-to-six months were required to test all 16 hues; thus, baseline results represent testing conducted for more than a year. As predicted, before treatment monkeys had low thresholds (averaging,0.03 units in u9, v9 colour space) for colours that represent blues and yellows to their eyes, but always failed to discriminate between the blue-green and the red-violet (dominant wavelengths of 490nm and 2499 nm, respectively) hues, with thresholds extrapolated from psychometric functions being orders of magnitude higher (Fig. 2b, c). Results were highly repeatable, with no improvement between the first and third tests, making us confident that the animals would not spontaneously improve in the absence of treatment.

Co-expressing the L-opsin transgene within a subset of endogenous M-cones shifted their spectral sensitivity to respond to long wavelength light, thus producing two distinct cone types absorbing in the middle-to-long wavelengths, as required for trichromacy. The spectral sensitivity shift was readily detected using a custom-built wide-field colour multifocal electroretinogram (mf-ERG) system (Fig. 1b, c, f) (see ref. 7 for details). In preliminary experiments, validity of the colour mf-ERG was tested using an animal that had received a mixture of the L-opsin-coding virus plus an identical virus, except that a green fluorescent protein (GFP) gene replaced the L-opsin gene. As reported previously, faint GFP fluorescence was first detected at 9 weeks post-injection, and it continued to increase in area and intensity over 24 weeks8. Although faint signs of GFP were first detectable at 9 weeks, L-opsin levels sufficient to produce suprathreshold mf-ERG signals were still not present at 16 weeks post-injection (Fig. 1c, inset). After GFP fluorescence became robust, the red light mf-ERG, which indicates responses from the introduced L-opsin, showed highly increased response amplitudes in two areas (Fig. 1c) corresponding to locations of subretinal injections.

The two dichromatic monkeys who participated in behavioural tests of colour vision were treated with L-opsin-coding virus only.

Although the elongated pattern produced by twoinjections allowed mf-ERG validation, the treatment goal was to produce a homogeneous region, as resulted from three injections shown in which the highest mf-ERG response covered about 80u of the central retina—roughly the area for which humans have good red–green discrimination. These results demonstrate that gene therapy changed the spectral sensitivity of a subset of the cones. A priori, there were two possibilities for how a change in spectral sensitivity might change colour vision behaviour. First, animals may have an increase in sensitivity to long-wavelength light, but if the neural circuitry for extracting colour information from the nascent 'M1L cone' submosaic was absent, they would remain dichromatic—the hallmark of which is having two hues that are indistinguishable from grey. The spectral neutral point for individuals that have only S and M cones (for example, monkeys 1 and 2 pre-therapy) occurs near the dominant wavelength of 495nm. At the limit, an increase in spectral sensitivity would shift themonkeys' neutral point towards that of individuals with only S and L cones, near the dominant  wavelength of 505nm. The second, more engaging possibility was that treatment would be sufficient to expand sensory capacity in monkeys, providing them with trichromatic vision. In this case, the animals' post-therapy results would appear similar, obtained from a trichromatic female control monkey.

Daily testing continued after treatment. After about 20 weeks postinjection the trained monkeys' thresholds for bluegreen and red-violet (dominant wavelengths of 490 and 2499 nm) improved, reducing to an average of 0.08 units in u9, v9 colour space, indicating that they gained trichromatic vision.

This time point corresponded to the same period in which robust levels of transgene expression were reported in the squirrel monkey8.

A trichromatic female monkey and untreated dichromatic monkeys were tested in parallel. As expected, the female had low thresholds for all colours, averaging ,0.03 units in u9, v9 colour space, but the untreated dichromats always failed to discriminate between dominant wavelengths of 490nm and 2499 nm, indicating a clear difference between treated and untreated monkeys.

Early experiments in which we obtained negative results served as 'sham controls', demonstrating that acquiring a new dimension of colour vision requires a shift in spectral sensitivity that results from expression of an L pigment in a subset of M cones. Using similar subretinal injection procedures, we delivered fewer viral particles of an L-opsin-coding rAAV2/5 virus with an extra 146-base-pair (bp) segment near the splice donor/acceptor site that had been carried over from the cloning vector and that was absent in the GFP-coding rAAV2/5 virus. The 146-bp segment contained an ATG and a duplicate messenger RNA start site that may have interfered with expression (see Methods). Three monkeys received injections of this vector, containing an average of 1.731012 virus particles per eye, and no reliable changes in spectral sensitivity were measured using the ERG.

One animal was also tested behaviourally and his colour vision was unchanged from baseline 1 year after injection. In subsequent experiments reported here, we removed the extra 146-bp segment and also increased the amount of viral particles delivered per eye by approximately 16-fold, to 2.731013. Negative results from earlier injections demonstrated that the subretinal injection procedure itself does not produce changes in the ERG or in colour vision.

The change in spectral sensitivity measured with the mf-ERG is necessary but not sufficient to produce a new colour vision capacity.

For example, individualswith L but noMcones (termed deuteranopes) have a relatively enhanced sensitivity to red light, but they are still as dichromatic as individuals withMbut no L cones (protanopes) in that they are unable to distinguish particular 'colours' from grey. To verify that the behavioural change observed in animals expressing the L pigment transgene was not purely a shift in spectral sensitivity, monkey 1 was also tested on dominant wavelengths of 496 and 500 nm, and monkey 2 was tested on dominant wavelengths of 496 and 507 nm. Together, these dominant wavelengths span the possible confusion points for deuteranopes and protanopes and for any intermediate dichromatic forms that could arise from expressing combinations of L and M pigments. Both monkeys' measured thresholds for these extra hues were similar to their thresholds for a dominant wavelength of 490 nm, demonstrating that they now lacked a spectral neutral point and have become truly trichromatic.

Furthermore, treated monkeys were able to discriminate blue-green (dominant wavelength of 490 nm) when it was tested against a redviolet (dominant wavelength of 2499 nm) background, instead of the grey background, indicating that the monkeys' newly-acquired 'green' and 'red' percepts were distinct from one another.The treatedmonkeys' improvement in colour vision has remained stable for more than 2 years and we plan to continue testing the animals to evaluate long-term treatment effects.

Classic experiments in which visual deprivation of one eye during development caused permanent vision loss1 led to the idea that inputs must be present during development for the formation of circuits to process them. From the clear change in behaviour associated with treatment, compared both between and within subjects, we conclude that adult monkeys gained new colour vision capacities because of gene therapy. These startling empirical results provide insight into the evolutionary question of what changes in the visual system are required for adding a new dimension of colour vision. Previously, it seemed possible that a transformation from dichromacy to trichromacy would require evolutionary/developmental changes, in addition to acquiring a third cone type. For example, L- and M-opsinspecific genetic regulatory elements might have been required to direct the opsins into distinct cone types9 that would be recognized by L- and M-cone-specific retinal circuitry10, and to account for cortical processing, multi-stage circuitry11 might have evolved specifically for the purpose of trichromacy. However, our results demonstrate that trichromatic colour vision behaviour requires nothing more than a third cone type. As an alternative to the idea that the new dimension of colour vision arose by acquisition of a new L versus M pathway, it is possible that it exploited the pre-existing blue-yellow circuitry. For example, if the addition of the third cone class split the formerly S versus M receptive fields into two types with differing spectral sensitivities, this would obviate the need for neural rewiring as part of the process of adopting new colour vision.

Some form of inherent plasticity in the mammalian visual system can be inferred from the acquisition of new colour vision, as was also demonstrated in genetically engineered mice12; however, the point has been made that such plasticity need not indicate that any rewiring of the neural circuitry has occurred13. Similarly, given the fact that new colour vision behaviour in adult squirrel monkeys corresponded to the same time interval as the appearance of robust levels of transgene expression, we conclude that rewiring of the visual system was not associated with the change from dichromatic to trichromatic vision.

Treated adultmonkeys unquestionably respond to colours that were previously invisible to them. The internal experiences associated with the marked change in discrimination thresholds measured here cannot be determined; therefore, we cannot knowwhether the animals experience new internal sensations of red and green. Nonetheless, we do know that evolution acts on behaviour, not on internalized experiences, and we suggest that gene therapy recapitulated what occurred during evolution of trichromacy in primates. These experiments demonstrate that a new colour-vision capacity, as defined by new discrimination abilities, can be added by taking advantage of pre-existing neural circuitry and, internal experience aside, full colour vision could have evolved in the absence of any other change in the visual system except the addition of a third cone type.

Gene therapy trials are underway for Leber's congenital amaurosis 14–16. Thus far, treatment has been administered to individuals who have suffered retinal degeneration from the disease. The experiments reported here are, to our knowledge, the first to use gene therapy in primates to address a vision disorder in which all photoreceptors are intact and healthy, making it possible to assess the full potential of gene therapy to restore visual capacities. Treatment allowing monkeys to see new colours in  adulthood provides a striking counter-example to what occurs under conditions of monocular deprivation. For instance, it is impossible to restore vision in an adult who had grown up with a unilateral cataract. Future technologies will allow many opportunities for functions to be added or restored in the eye. Although some changes may produce outcomes analogous to monocular deprivation, we predict that others, like gene therapy for red–green colour blindness, will provide vision where there was previously blindness.