NZ735735B2 - Gene therapy to improve vision - Google Patents

Abstract

The invention relates to the use of gene therapy vectors to improve vision by introducing into healthy rod photoreceptor cells of a patient suffering from cone photoreceptor dysfunction and/or degeneration a nucleic acid encoding a gene product that is light-sensitive and/or that modulates endogenous light-sensitive signalling in a photoreceptor cell, such that the range of light intensities to which the rod photoreceptor responds is extended and/or the speed at which the rod photoreceptor responds to light is increased. In a particular embodiment, the gene product is ArchT or R9AP, and the capsid of the vector is derived from AAV8. s light-sensitive signalling in a photoreceptor cell, such that the range of light intensities to which the rod photoreceptor responds is extended and/or the speed at which the rod photoreceptor responds to light is increased. In a particular embodiment, the gene product is ArchT or R9AP, and the capsid of the vector is derived from AAV8.

Description

GENE THERAPY TO IMPROVE VISION FIELD OF THE INVENTION The present invention relates to the use of gene therapy vectors to improve vision in patients.

BACKGROUND OF THE INVENTION In many mammalian species including mice and humans, the number of rod photoreceptors that mediate vision under dim light outnumbers greatly that of cone photoreceptors (Curcio et al, 2000). However, in an industrialised world where illumination allows cones to operate throughout the day, rod-mediated vision is less important. Many patients with absent rod function from birth are identified only incidentally and, in fact, cannot recognize their abnormal vision (Dryj a, 2000). On the contrary, when cone ction is present, patients are always symptomatic and often suffer visual handicap dependent on the degree of their cone ction. In some conditions, however, only (or ) the cones are lost or dysfunctional and rods remain relatively preserved. For example, achromatopsia is a severe hereditary retinal phy with a complete absence of cone function from birth but, presumably, with a normal rod fiinction (Hess et al, 1986; Nishiguchi et al, 2005). Mutations in multiple genes including CNGA3 (Kohl et al 1998), and PDE6C (Chang et al, 2009, Thiadens et al, 2009) have been associated with the disease. Each of the e causing genes encodes an essential component of the cone phototransduction cascade that translates light into an electric signal by causing hyperpolarization of the photoreceptor cell. In age d macular degeneration (AMD), visual impairment is caused primarily by degeneration of the cone-rich fovea in the central macula. Thus ts lose central vision and acuity, but often have relatively well preserved peripheral macula and thus have some useful residual vision that is d by the paucity of cones outside the fovea.

Rods are highly sensitive to light, which s them to perceive a small amount of light in dim conditions. Cones, on the contrary, are less sensitive, but are capable of processing large amounts of light and uously convey visual signals in daylight.

This fiinctional difference is, in part, due to the efficiency of the deactivation ery of photo-signalling, the GTPase complex composed of RGS9, R9AP (also known as RGS9BP), and G85. RGS9 is the catalytic component that hydrolyses the GTP coupled to the G-protein, whereas R9AP and G135 are the essential constitutive ts (Burns et al, 2009; Burns et al, 2010). Importantly, R9AP tethers the complex to the disc membrane at the photoreceptor outer segment where the phototransduction signalling also takes place (Baseler et al, 2002). Expression of R9AP determines the level of GTPase complex such that any RGS9 produced in excess of R9AP is likely quickly degraded (Martyemyanov et a1, 2009). Over-expression of R9AP in the murine rods is sufficient to increase the GTPase activity and to substantially speed their deactivation cs as evidenced by the single cell recordings (Krispel et al, 2006). In the cones, the RGS9 expression has been estimated to be ~10-fold higher than that of the rods (Cowan et a1, 1998; Zhang et al, 2003). This provides a basis for the ability ofthe cones to recover quickly from light exposure and thus maintain functional to continuous light stimulus. It also allows cones to respond to more rapid stimulation. Indeed, clinically, patients with delayed deactivation of phototransduction cascade caused by genetic defects in RGS9 or R9AP, or bradyopsia, have a nd impairment ofcone-mediated vision including day blindness and d y to see moving objects Nishiguchi et a1, 2004; Michaelides et al, 2010). ile, the rod-mediated vision is less ed by the same mutation.

Some macular ration conditions, such as age-related macular degeneration (AND) and inherited macular degeneration conditions also exhibit cone dysfunction but normal or less impaired rod function. Macular ration is the leading cause of blindness in the developed world and as its prevalence quadruples in each decade of life the instance of AMD is expected to rise in the coming years as life expectancy increases. Drugs for the treatment of AMD already account for over 1% of the entire drugs budget of the UK’ s al Health e. While patients with advanced AMD can be trained to fixate extra-foveally, the low refresh rate and the low bleaching threshold of rod cells limits the quality of resulting vision.

SUMMARY OF THE INVENTION Using mice with absent cone function, we have demonstrated that AAV-mediated overexpression of Rgs9-anchor protein (R9AP), a critical component of GTPase complex that mediates the deactivation of phototransduction cascade, results in desensitization and “photopic shift” ofthe rod-driven oretinogram. The treatment enables the rods to respond to brighter light (up to ~20 log) than the ted cells at the expense of scotopic (lower light level) fiinction. Multi-electrode array measurements using the treated retinas showed that the retinal ganglion cells also reflected the “photopic shift” of the rods, by exhibiting graded responses at ic light levels. Contrast sensitivity function measured by quantifying the racking movements in response to rotating sinusoidal gs showed an ement of the sensitivity by up to 25-fold under room light conditions and faster response to repeated stimuli. Furthermore, biochemical measurement of bleachable rhodopsin levels in these mice indicated that the visual cycle was not limiting rod function.

We have also expressed a fast light-driven proton pump, ArchT (Han et al, 2011) in rod photoreceptors. AAV8 particles carrying ArchT-EGFP under control of the Rhodopsin promoter (Rho) were ed subretinally in adult mice. Expression of Rho-ArchT- EGFP was limited to the membrane of rod photoreceptors. Expression of ArchT allowed extremely rapid light responses, while the intrinsic rod response was preserved and was comparable to that observed in non—transduced rod photoreceptors. Overall, ArchT expression did not alter the y of rod photoreceptors to respond to scotopic stimuli, but it did confer an additional ability to respond with rapid non-bleaching responses to higher levels of illumination. We also found that the transduced rods were able reliably to sustain this fast ‘cone-like’ transmission, in that ArchT-expressing rods drove sustained retinal on cell (RGC) spiking at high light intensities and at frequencies approaching those of cone photoreceptors, Expression of Rho-ArchT- EGFP in CNGA3-/- and PDE6C-/- mice lacking cone-mediated vision also extended the sensitivity of these mice to bright light stimuli and conferred fast vision on these mice. The maximal frequency of stimuli that ArchT-expressing mice could follow was similar to that of cone photoreceptors.

Together, these results show that, after transduction of y rod photoreceptors with genes encoding either light sensitive proteins characteristic of cones or genes encoding molecules that increase the speed of the endogenous rod signalling mechanism, rods behave more like cones and hence can compensate for cone ction. This has implications for the treatment of a number of vision disorders in which cone function is reduced, but at least some healthy rods remain. This contrasts with previous ches (Busskamp et al, 2010; US Patent Publication No. 2012258530) in which the goal was to restore lost on in cones. Altering function in rods in the manner of the present invention is advantageous in that conditions in which cones are dysfunctional but can be repaired (for example in the early stages of retinal ration when photoreceptor function is lost but the photoreceptor-to-bipolar synapse may be intact) are rare, whereas ions in which cone dysfunction is more severe or advanced and cannot be repaired or where cones are lost entirely, but yet at least some healthy rod photoreceptors remain, are common (see above). Furthermore, this ion enables the on of a ‘pseudo-fovea’, a small patch of cone-like rods that will improve vision in conditions in which foveal cones have been lost or are dysfunctional.

The ion therefore provides a vector sing a nucleic acid encoding a gene product that is light-sensitive and/or that modulates endogenous light-sensitive signalling in a photoreceptor cell, for use in a method of improving vision in a patient with cone photoreceptor dysfunction and/or degeneration by introduction of said nucleic acid into healthy rod photoreceptors in the retina of the patient and expression of said gene product therein, such that the range of light intensities to which the rod photoreceptor responds is extended and/or the speed at which the rod photoreceptor responds to light is increased.

The invention also provides a vector as defined above, a host cell comprising said vector and methods of treatment carried out with such a .

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Expression of ArchT in rod photoreceptors leads to fast light-driven currents. (a), top panels: ediated transduction of ArchT—EGFP (green, see left ) under control of the Rhodopsin promoter. No overlap is observed with cones (purple: Cone arrestin, see middle panels, white: DAPI, see right panels). Lower panels: specificity of expression can also be observed at the level of synapses. (b) ArchT-EGFP is localized to the ne of rod photoreceptors, including inner and outersegments. (c) Quantification of fluorescence in EGFP-expressing rod als (green, left peak) and Cone in positive cone terminals (purple, right peak) shows two ct bands corresponding to the sub-layer where rod and cone synapses localize respectively (n=22). (d) Single-cell recordings from the cell bodies of ArchT-expressing rod photoreceptors. The currents mediated by the intrinsic rod photo-trandsuction (upper trace) in response to 10 ms 530-nm light pulses (green, see vertical bars) were preserved. The ArchT-generated currents were faster (lower . Scale bars: (a) upper panels: 50 um, (a) lower panels and (b): 10 um.

Figure 2: ArchT-expression drives high-frequency responses in rods and fast transmission to Retinal Ganglion Cells. 2016/050419 (a) Intrinsic rod light-evoked currents in uninjected C57BL6 retinas. (b) ArchT- mediated currents are able to follow much higher stimulation ncies. (c) Responses are time-locked to stimulus presentation (green vertical bars). ((1) ArchT- expressing rods respond to frequency stimulation up to 80 Hz t faltering, whereas intrinsic rod responses drop off at ~20 Hz. (e) y data showing that ArchT expression does not alter the intrinsic response of rod photoreceptors, while ArchT responses begin at brighter light levels. (f) Multi-electrode array recordings from PDE6C-/- ArchT-expressing retinas. sic rod responses failed to elicit reliable Retinal Ganglion Cell spiking above 20 Hz.. On the contrary, ArchT-mediated rod activation drove fast g of Retinal Ganglion Cells to levels able to cone photoreceptors.

Figure 3: ArchT-mediated activation of rods drives behavioural response for fast high light intensity-stimuli. (a) Top panels: schematic for fear conditioning behaviour. Briefly, a visual stimulus was paired with a shock. 24 hours later freezing behaviour was tested in a new context.

Bottom : uninjected CNGA3'/' and /' mice failed to learn the task (left sets of bars in each graph). However, ArchT expression successfully drove freezing behaviour in mice (right sets of bars in each graph). (b) Optomotor testing. ArchT- expressing mice are able to follow stimuli at frequencies comparable to those reliably followed by cones.

A. Figure 4: diated R9AP over-expression in rods and accelerated a- wave deactivation in /- mice.Increased RGS9 expression in a Cnga3-/- eye treated with rRAAV2/8.Rho.mR9ap. Over-expression of R9AP results in increased immunoreactivity toward RGS9 (red) throughout the photoreceptor layer in the treated eye (left) compared to the untreated (right). Western blot shows increased expression of RGS9 both in the retina and retinal pigment epithelium (RPE) in the eye over- expressing R9AP (bottom). A small amount of RGS9 protein was also detected in the WO 35457 RPE of the treated eye. This may reflect “spill over” of the excessive protein contained within the phagocytosed disc membrane. Scale bar tes 25 um.

B. Increased speed of a-wave amplitude ry in the Cnga3-/— eye treated with rAAV2/8.Rho.mR9ap and rAAV2/8.CMV.mR9ap. Representative ERG tracings for the probe flash (black traces, see traces with peak in the middle of the timecourse) and for the 2nd flash (red traces) presented at inter-stimulus interval (181) of2 s from the treated (top) and untreated (bottom) eyes from the same animal. Note that a second flash yields small a-wave (arrow) is clearly visible in the treated eye, whereas a-wave is not visible (arrow) in the untreated fellow eye. A plot of a-wave recovery at various ISIS in the treated and untreated eyes. The eyes injected with rAAV2/8.CMV.mR9ap (n = 5) or rAAV2/8.Rho.mR9ap (n = 7) have faster recovery kinetics than the ted eye (11 = 5) that is most visible with shorter ISIs. The data is presented as average :: standard error of the mean. OE: over-expression.

Figure 5: Gain of photopic function by rods h over-expression of R9AP in Cnga3-/- mice.

A. Elevation of se threshold and photopic shift of 6H2 ERGs through over- expression of R9AP in rods of Cnga3-/- mice. Representative 6H2 ERG traces from a Cnga3-/— mouse in which one eye was treated with rAAV2/8.CMV.mR9ap and the other eye was left untreated (top panel). ERG traces are aligned from responses against the dimmest flash (-6.0 log eds/ml) to the brightest flash (2.0 log eds/ml, bottom) from the top to the bottom at 0.5 log.cd.s/m2 step. Note that the lower threshold flash intensity at which the responses emerge is increased, which is coupled with elevated response threshold to brighter . This results in a “photopic shift” of the retinal function in the eye treated with rAAV2/8.CMV.mR9ap. Summary of 6H2 ERG results demonstrating photopic shift of the retinal function following treatment with rAAV2/8.CMV.mR9ap or rAAV2/8.Rho.mR9ap m panel). ERG ses from Gnat1-/- mice nt in rod function represents cone-mediated function. Meanwhile, responses from C57BL6 mice are derived from both rod and cone photoreceptors. The 2016/050419 data is presented as % amplitude relative to the maximal response and is presented as average :: standard error of the mean. ERGs were recorded from Cnga3-/- mice treated with rAAV2/8.CMV.mR9ap (Cnga3 -/- CMV.R9ap; N = 8), Cnga3-/- mice treated with rAAV2/8.Rho.mR9ap (Cnga3-/- Rho.R9ap; N = 6), Cnga3-/— mice untreated (Cnga3- /- Untreated; N = 8), Gnat1-/- mice ted (Gnatl-/- ted; N = 6), and C57BL6 mice untreated (C57BL6 Untreated; N = 6) B. sed retinal responses to long flashes in the Cnga3-/— eye treated with rAAV2/8.CMV.mR9ap. Open rectangle denotes the duration of the flash. Note that in the eye treated with rAAV2/8CMVmR9ap, responses are detectable with increased duration of light stimulus. Conversely, the untreated contralateral eye shows little or no response when recorded simultaneously at identical conditions.

C. Gain of retinal on under photopic conditions in the Cnga3-/- eyes treated with rAAV2/8.CMV.mR9ap. Note that the treated eye shows responses under photopic ing ions (white background light of 20 cd/mz) whereas the untreated contralateral eye recorded simultaneously remained unresponsive.

Figure 6: Efficient transmission of the altered photoreceptor signal to the bipolar cells in the eyes over-expressing R9AP A. Representative ERG . ERGs were recorded after rAAV2/8.CMV.mR9ap injection (red trace, lower trace) in a Cnga3-/— mouse using a saturating flash (1.9 log cd.s/m2). The contralateral eye served as an untreated control (black trace, upper trace).

B. d activation of the bipolar cells to a flash. A-wave and b-wave implicit times were measured from an ERG response to a saturating flash (1.9 log cd.s/m2) in the treated and untreated eyes.

C. Intensity se curve for a—wave and b-wave amplitudes recorded from Cnga3-/- mice (N=5) with one eye treated with rAAV2/8.CMV.mR9ap (red curves) and the other eye left untreated (black curves). All data are presented as average :: standard error of the mean. OE: over-expression. 2016/050419 Figure 7: A gain of sustainable visual perception following R9AP over-expression in Cnga3-/- mice.

A. Improved contrast sensitivity function measured by optokinetic responses. In the Cnga3-/- mice treated with 8.Rho.mR9ap in the left eye, the contrast sensitivity function (CSF) was differentially measured for clockwise (representing treated left eye) and counter-clockwise (representing untreated right eye) head tracking movements against sinusoidal gratings. The CSF for the treated eyes (red curve) was better than that for the untreated eyes (blue , which was similar to that for the hed Cnga3 -/- mice (black curve; average ofboth eyes). Note that the CSF for the treated eye was equivalent to, if not slightly better, to that for untouched wild—type controls (green; average of both eyes). N= 5 for all groups. All data are ted as average it standard error of the mean. OE: xpression.

B. Sustained rhodopsin levels after prolonged exposure to Optomotry test. A representative recording of optical absorption of ocular sample using ng spectrophotometer (left panel inside the green dashed box). Subtracting the absorption of ocular s measured after (blue trace, top trace between 300 and 400nm) from before (red trace) complete photobleaching showed )tmin peaking at ~3 80 nm corresponding to released photoproducts coupled with kmax peaking at ~500 nm representing the amount of bleachable rhodopsin in the sample (right panel inside the green dashed box). Rhodopsin bleaching speed was assessed by measuring the difference um (kmax) in the fully dilated eyes treated or untreated with rAAV2/8.Rho.mR9ap in Cnga3 -/- mice after 5 minutes’ exposure to 7.0 mW white light (bottom left; average :: standard error of the mean). Rhodopsin levels was measured also after exposure of the Cnga3-/— mice to Optomotry test for up to 120 s (right bottom, N=3 for each time point) following unilateral injection of rAAV2/8.Rho.mR9ap. The grey area tes rhodopsin levels (mean i standard deviation) recorded from untouched Cnga3-/— mice (N=8) after an overnight dark- adaptation. Dotted line tes the average. Note that the level of rhodopsin remains stable for at least 2 hours in both the eyes treated with rAAV2/8.Rho.mR9ap and the ted eyes ofCnga3-/— mice. Data with error bars were presented as mean ::standard error of the mean.

Figure 8: Over-expression of R9AP increases the recovery speed of rod photoresponse in /- mice.

The time constant (c) for 50% recovery of a-wave amplitude was reduced by ~50% in the Pde6c-/- eyes ed with rAAV2/8.CMV.mR9ap (o = ~ 5.75 sec) compared to the untreated contralateral eyes (6 = ~ 11.46 sec) consistent with accelerated deactivation of phototransduction following the treatment. N = 6. Data with error bars were displayed as mean ::standard error of the mean.

Figure 9: Over-expression of R9AP s in “photopic shift” of the intensity- response curve in Pde6c-/- mice.

The eyes injected with rAAV2/8.CMV.mR9ap showed photopic shift ofthe 6 Hz ERG responses to ental flash intensities compared to the untreated contralateral eyes in Pde6c-/- mice (N = 6). The data is presented as % amplitude relative to maximal response and is displayed as e i standard error of the mean.

Figure 10: Sustained effect of R9AP over-expression without clear evidence of retinal ration at 5 months post-injection of rAAV2/8.CMV.mR9ap in Cnga3-/- mice.

Response profile normalized against maximum amplitude confirmed the presence of “photopic shift” of the intensity-response curve to 6Hz flashes (top). The same data without normalization showed no evidence of reduction in amplitudes in the treated eye m). N = 5. The data is presented as average :: standard error of the mean.

Figure 11: Treatment of wild-type mice with rAAV2/8.Rho.mR9ap showed no obvious effect on 6 Hz ERG ity-response curve.

The eyes treated with rAAV2/8.Rho.mR9ap showed no shift in 6 Hz ERG intensity- response curve compared to that for the untreated contralateral eyes in C57BL6 mice (N = 5). The data is presented as average :: standard error of the mean.

Figure 12: No gain of visual perception following R9AP xpression in C57BL6 mice.

In the C57BL6 mice treated with rAAV2/8.Rho.mR9ap only in the left eye, the contrast sensitivity function (CSF) was differentially ed for ise (representing treated left eye) and counter-clockwise (representing untreated right eye) head tracking movements to rotating sinusoidal gratings. The CSF for both the treated eyes (pink curve) and the untreated eyes (light blue curve) showed similar results, which was similar to that for the untouched C57BL6 mice (green curve; average ofboth eyes). N= for all groups. All data are presented as average :: standard error of the mean. OE: over-expression.

DETAILED DESCRIPTION OF THE INVENTION A vector of the invention comprises a c acid whose expression to e a gene product, typically a protein, which will effect treatment of an ocular condition as described herein, operably linked to a promoter to form an expression cassette.

Nucleic Acids and Gene Products A vector ofthe ion comprises a nucleic acid encoding a gene product that is light- sensitive and/or that modulates endogenous light-sensitive signalling in a photoreceptor cell and makes a rod transduced with the nucleic acid of the invention behave more like a cone by extending the range light intensities to which the rod eceptor responds is and/or increasing the speed at which the rod photoreceptor responds to light. Thus, the n may itself be directly light-sensitive, e.g. it may change ne conductance in rods in a way that results in hyperpolarisation (outward t flow) upon light stimulation. Such proteins will for example be light-sensitive or light-gated G—coupled membrane proteins, ion channels, ion pumps or ion transporters. Preferred light-sensitive proteins include ArchT, Jaws (cruxhalorhodopsin) (Chuong et al, 2014) and iClC2. atively, the protein may itself not be directly light-sensitive but may indirectly modulate endogenous light-sensitive signalling in a rod photoreceptor cell.

Examples of such proteins are members of the RG89 complex, notably R9AP (also known as RGS9BP), and G135. In the ative, the nucleic acid may encode any other gene product that increases the speed of the endogenous rod signalling mechanism. In all of these cases, the sequence may encode a wild-type protein or a mutant or variant or truncated version that retains the activity of the wild—type protein. The nucleic acid may also be codon-optimised for expression in the target cell type.

Following expression of the gene t, rods will show stronger and/or faster modulation to light stimuli than non—transduced rods, at higher than usual intensities.

Examples e improved modulation strength and/or faster activation/inactivation kinetics. Rods transduced according to the invention will therefore react more strongly and/or quickly to illumination in the mesopic and/or photopic range than non- transduced rods. Preferably, the response of the rods to scotopic illumination conditions is cted or not substantially affected, ie the rods gain the ability to respond strongly and/or quickly to brighter light without losing the ability to d to dim light.

Promoters and other regulatory elements In the expression construct, the nucleic acid encoding the gene t is typically operably linked to a promoter. The promoter may be constitutive but will preferably be a photoreceptor-specific or photoreceptor-preferred promoter, more preferably a rod—specific or rod-preferred promoter such as a Rhodopsin (Rho), Neural retina- specific leucine zipper protein (NRL) or Phosphodiesterase 6B (PDE6B) promoter. The promoter region incorporated into the expression cassette may be of any length as long as it is effective to drive sion of the gene product, preferably photoreceptor- specific or photoreceptor-preferred expression or rod-specifrc or rod- preferred expression.

By a eceptor-specific promoter, is meant a promoter that drives expression only or substantially only in photoreceptors, e.g. one that drives expression at least a hundred-fold more strongly in photoreceptors than in any other cell type. By a rod- specifrc promoter, is meant a promoter that drives expression only or ntially only in photoreceptors, e. g. one that drives sion at least a hundred-fold more strongly in photoreceptors than in any other cell type, including cones. By a photoreceptor- preferred promoter, is meant a promoter that expresses preferentially in photoreceptors but may also drive expression to some extent in other s, e.g. one that drives expression at least two-fold, at least five-fold, at least ten-fold, at least 20-fold, or at least d more strongly in photoreceptors than in any other cell type. By a rod- preferred promoter, is meant a promoter that drives sion preferentially in photoreceptors but may also drive expression to some extent in other tissues, e.g. one that drives expression at least two-fold, at least five-fold, at least ten-fold, at least 20- fold, or at least 50-fold more strongly in photoreceptors than in any other cell type. including cones.

One or more other regulatory ts, such as enhancers, may also be present as well as the promoter.

Vectors A vector of the invention may be of any type, for example it may be a plasmid vector or a minicircle DNA.

Typically, vectors of the invention are however viral vectors. The viral vector may for example be based on the herpes x virus, adenovirus or lentivirus. The viral vector may be an adeno-associated virus (AAV) vector or a derivative thereof. The viral vector derivative may be a chimeric, shuffled or capsid modified tive.

The viral vector may comprise an AAV genome from a naturally derived serotype, isolate or clade of AAV. The serotype may for example be AAV2, AAVS or AAV8.

The efficacy of gene y is, in general, dependent upon te and efficient delivery ofthe donated DNA. This process is usually mediated by viral vectors. Adeno- associated viruses (AAV), a member of the parvovirus family, are commonly used in gene therapy. Wild-type AAV, containing viral genes, insert their genomic material into chromosome 19 of the host cell. The AAV single-stranded DNA genome comprises two ed terminal s (ITRs) and two open reading frames, containing structural (cap) and packaging (rep) genes.

For therapeutic purposes, the only sequences required in cis, in addition to the therapeutic gene, are the ITRs. The AAV virus is therefore modified: the viral genes are removed from the genome, producing recombinant AAV (rAAV). This contains only the therapeutic gene, the two ITRs. The removal of the viral genes renders rAAV incapable of actively inserting its genome into the host cell DNA. d, the rAAV genomes fuse via the ITRs, forming circular, episomal structures, or insert into pre- existing somal breaks. For viral production, the structural and packaging genes, now removed from the rAAV, are supplied in trans, in the form of a helper plasmid.

AAV is a ularly attractive vector as it is lly non-pathogenic; the majority people have been infected with this virus during their life with no adverse effects.

The immune privilege of ocular tissue, a result of anatomical barriers and immunomodulatory s, renders the eye largely exempt from the adverse immunological responses that can be triggered in other tissues by AAV (Taylor 2009).

AAV vectors are limited by a relatively small packaging ty of roughly 4.8kb and a slow onset of expression following transduction. Despite these minor drawbacks, AAV has become the most ly used viral vector for retinal gene therapy.

Most vector constructs are based on the AAV serotype 2 (AAV2). AAV2 binds to the target cells Via the heparin sulphate proteoglycan receptor. The AAV2 genome, like those of all AAV serotypes, can be enclosed in a number of different capsid proteins.

AAV2 can be ed in its natural AAV2 capsid (AAV2/2) or it can be pseudotyped with other capsids (eg. AAV2 genome in AAVl capsid; AAV2/ 1, AAV2 genome in AAV5 , AAV2/5 and AAV2 genome in AAV8 capsid, AAV2/8). rAAV transduces cells via serotype specific receptor-mediated endocytosis. A major factor influencing the cs of rAAV transgene expression is the rate of virus particle uncoating within the endosome. This, in turn, depends upon the type of capsid enclosing the genetic material (Ibid.). After uncoating the linear single-stranded rAAV genome is stabilised by forming a -stranded molecule via de novo synthesis of a complementary strand. The use of self-complementary DNA may bypass this stage by producing double-stranded transgene DNA. Natkunaraj ah et al (2008) found that selfcomplementary AAV2/8 gene expression was of faster onset and higher amplitude, ed to single-stranded . Thus, by circumventing the time lag associated with second-strand synthesis, gene expression levels are increased, when compared to transgene expression from standard single-stranded ucts. Subsequent studies investigating the effect of self-complementary DNA in other AAV pseudotypes (e. g.

AAV2/5) have produced similar results. One caveat to this technique is that, as AAV has a packaging capacity of approximately 4.8kb, the self-complementary inant genome must be appropriately sized (i.e. 2.3kb or less).

In addition to modifying packaging capacity, pseudotyping the AAV2 genome with other AAV capsids can alter cell specificity and the cs of transgene expression.

AAV2/8 is reported to transduce photoreceptors more efficiently than either AAV2/2 or AAV2/5 (Natkunaraj ah et al. 2008).

The vector of the invention may therefore se an adeno-associated virus (AAV) genome or a derivative thereof.

An AAV genome is a polynucleotide sequence which s functions needed for production of an AAV viral particle. These functions include those operating in the replication and packaging cycle for AAV in a host cell, including encapsidation of the AAV genome into an AAV viral particle. Naturally occurring AAV viruses are replication-deficient and rely on the ion of helper functions in trans for completion of a replication and packaging cycle. Accordingly and with the onal removal of the AAV rep and cap genes, the AAV genome ofthe vector ofthe invention is ation-deficient.

The AAV genome may be in single-stranded form, either ve or negative-sense, or alternatively in double-stranded form. The use of a double-stranded form allows bypass ofthe DNA replication step in the target cell and so can accelerate transgene expression.

The AAV genome may be from any naturally derived serotype or isolate or clade of AAV. As is known to the skilled person, AAV viruses occurring in nature may be fied according to various biological systems. ly, AAV viruses are referred to in terms of their serotype. A serotype corresponds to a variant subspecies ofAAV which owing to its profile of expression of capsid surface antigens has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, a virus having a particular AAV serotype does not efficiently cross-react with neutralising antibodies specific for any other AAV serotype. AAV serotypes include AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVlO and AAVl 1, also recombinant serotypes, such as Rec2 and Rec3, ly identified from primate brain. In vectors of the invention, the genome may be derived from any AAV serotype. The capsid may also be derived from any AAV serotype. The genome and the capsid may be derived from the same serotype or different serotypes.

In vectors ofthe invention, it is preferred that the genome is derived from AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), AAV serotype 5 (AAVS) or AAV serotype 8 (AAV8). It is most preferred that the genome is derived from AAV2 but other serotypes of particular interest for use in the invention include AAV4, AAVS and AAV8, which efficiently transduce tissue in the eye, such as the retinal pigmented epithelium. It is red that the capsid is derived from AAVS or AAV8, especially AAV8.

Reviews of AAV serotypes may be found in Choi et a1 (Curr Gene Ther. 2005; 5(3); 299—310) and Wu et al (Molecular Therapy. 2006, 14(3), 316-327). The ces of AAV genomes or of elements of AAV genomes including ITR sequences, rep or cap genes for use in the invention may be derived from the following accession numbers for AAV whole genome sequences: Adeno-associated virus 1 NC_002077, AF063497 ; Adeno-associated virus 2 NC_001401; Adeno-associated virus 3 729, Adeno- associated virus 3B NC_001863; Adeno-associated virus 4 NC_001829; Adeno- associated virus 5 Y18065, 16; Adeno-associated virus 6 NC_001862, Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-l NC_006263, 83, Bovine AAV NC_005889, AY3 88617.

AAV viruses may also be referred to in terms of clades or . This refers to the phylogenetic relationship of naturally derived AAV viruses, and typically to a enetic group of AAV s which can be traced back to a common ancestor, and includes all descendants thereof. onally, AAV viruses may be referred to in terms of a specific isolate, i.e. a genetic isolate of a specific AAV virus found in nature.

The term genetic isolate describes a population of AAV Viruses which has undergone limited genetic mixing with other naturally occurring AAV Viruses, thereby defining a recognisably distinct population at a genetic level.

Examples of clades and isolates of AAV that may be used in the ion include: Clade A: AAVl NC_002077, AFO63497, AAV6 NC_001862, Hu. 48 AY530611, Hu 43 AY530606, Hu 44 AY530607, Hu 46 AY530609 Clade B: Hu. 19 84, Hu. 2O AY530586, Hu 23 AY530589, Hu22 AY530588, Hu24 AY530590, Hu21 AY530587, Hu27 AY530592, Hu28 AY530593, Hu 29 AY530594, Hu63 AY530624, Hu64 AY530625, Hu13 AY530578, Hu56 AY530618, Hu57 19, Hu49 AY530612, Hu58 AY530620, Hu34 AY530598, Hu35 AY530599, AAV2 NC_001401, Hu45 AY530608, Hu47 AY530610, Hu51 AY530613, Hu52 AY530614, Hu T41 AY695378, Hu S17 AY695376, Hu T88 AY695375, Hu T71 AY695374, Hu T70 AY695373, Hu T40 72, Hu T32 AY695371, Hu T17 AY695370, Hu LG15 AY695377, Clade C: Hu9 29, HulO 76, Hull AY530577, Hu53 AY530615, Hu55 AY530617, Hu54 AY530616, Hu7 AY530628, Hu18 AY530583, Hu15 AY530580, Hu16 AY530581, Hu25 AY530591, Hu60 AY530622, Ch5 AY243021, Hu3 AY530595, Hul AY530575, Hu4 AY530602 Hu2, AY530585, Hu61 23 Clade D: Rh62 AY530573, Rh48 61, Rh54 AY530567, Rh55 AY530568, Cy2 AY243020, AAV7 AF513851, Rh35 AY243000, Rh37 AY242998, Rh36 AY242999, Cy6 AY243016, Cy4 AY243018, Cy3 AY243019, Cy5 AY243017, Rh13 AY243013 Clade E: Rh38 AY530558, Hu66 AY530626, Hu42 AY530605, Hu67 AY530627, Hu40 AY530603, Hu41 AY530604, Hu37 AY530600, Rh40 AY530559, Rh2 AY243007, Bbl AY243023, Bb2 AY243022, Rth AY243015, Hu17 AY530582, Hu6 AY530621, Rh25 57, Pi2 AY530554, Pil AY530553, Pi3 AY530555, Rh57 AY530569, Rh50 AY530563, Rh49 AY530562, Hu39 AY530601, Rh58 AY530570, Rh6l AY530572, Rh52 AY530565, Rh53 AY530566, RhSl AY530564, Rh64 AY530574, Rh43 AY530560, AAV8 AF513852, Rh8 AY242997, Rhl AY530556 Clade F: Hul4 (AAV9) AY530579, Hu3l AY530596, Hu32 AY530597, Clonal Isolate AAVS Y18065, AF085716, AAV 3 NC_001729, AAV 3B NC_001863, AAV4 829, Rh34 AY243001, Rh33 AY243002, Rh32 AY243003/ The skilled person can select an appropriate serotype, clade, clone or e of AAV for use in the present invention on the basis of their common general knowledge. It should be understood however that the invention also encompasses use of an AAV genome of other serotypes that may not yet have been identified or characterised. The AAV serotype determines the tissue specificity of infection (or tropism) of an AAV virus. Accordingly, preferred AAV serotypes for use in AAV viruses administered to patients in accordance with the invention are those which have natural tropism for or a high efficiency of ion of rod photoreceptors.

Typically, the AAV genome of a naturally derived serotype or e or clade of AAV comprises at least one inverted terminal repeat sequence (ITR). Vectors ofthe ion typically comprise two ITRs, preferably one at each end of the genome. An ITR sequence acts in cis to provide a fiinctional origin of replication, and allows for integration and excision of the vector from the genome of a cell. Preferred ITR sequences are those of AAV2 and variants thereof. The AAV genome typically comprises packaging genes, such as rep and/or cap genes which encode packaging ons for an AAV viral particle. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid ns such as VPl, VP2 and VP3 or ts thereof. These proteins make up the capsid of an AAV viral particle. Capsid variants are discussed below.

Preferably the AAV genome will be derivatised for the e of stration to patients. Such tisation is standard in the art and the present invention encompasses the use of any known derivative of an AAV , and tives which could be generated by applying ques known in the art. Derivatisation of the AAV genome and of the AAV capsid are reviewed in, for example, Choi et al and Wu et al, nced above.

Derivatives of an AAV genome include any truncated or d forms of an AAV genome which allow for sion of a Rep-l transgene from a vector of the invention in viva. Typically, it is possible to truncate the AAV genome significantly to e minimal viral sequence yet retain the above function. This is preferred for safety reasons to reduce the risk of ination of the vector with wild-type virus, and also to avoid triggering a ar immune response by the presence ofviral gene proteins in the target cell.

Typically, a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR. A preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication ofthe genome to generate a single-stranded genome which ns both coding and complementary sequences i.e, a self-complementary AAV genome. This allows for bypass ofDNA replication in the target cell, and so enables accelerated transgene expression.

The one or more ITRs will preferably flank the expression construct cassette containing the promoter and transgene of the invention. The inclusion of one or more ITRs is preferred to aid packaging of the vector ofthe invention into viral particles. In preferred embodiments, ITR elements will be the only sequences retained from the native AAV genome in the derivative. Thus, a derivative will preferably not include the rep and/or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence ts (such as regulatory elements) within the vector in on to the transgene.

With reference to the AAV2 genome, the following portions could therefore be removed in a derivative of the invention: One inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes. However, in some embodiments, including in vitro embodiments, derivatives may additionally include one or more rep and/or cap genes or other viral sequences of an AAV genome.

A derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAV viruses. The invention encompasses the provision of capsid protein ces from ent serotypes, clades, clones, or isolates of AAV within the same vector. The invention encompasses the packaging of the genome of one serotype into the capsid of another serotype i.e. pseudotyping.

Chimeric, shuffled or capsid-modified derivatives will be typically selected to provide one or more desired fiinctionalities for the viral vector. Thus, these derivatives may display increased ncy of gene delivery, decreased immunogenicity (humoral or cellular), an d tropism range and/or improved targeting of a ular cell type compared to an AAV viral vector sing a naturally ing AAV genome, such as that of AAV2. Increased efficiency of gene delivery may be effected by improved receptor or co-receptor binding at the cell surface, improved internalisation, improved trafficking within the cell and into the nucleus, improved uncoating of the viral particle and improved sion of a single-stranded genome to -stranded form.

Increased efficiency may also relate to an altered tropism range or targeting of a specific cell population, such that the vector dose is not diluted by administration to tissues where it is not needed. ic capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This may be performed for example by a marker rescue ch in which non-infectious capsid sequences of one serotype are cotransfected with capsid sequences of a different serotype, and directed selection is used to select for capsid sequences having d properties. The capsid sequences of the different serotypes can be altered by homologous recombination within the cell to produce novel chimeric capsid proteins.

Chimeric capsid proteins also include those generated by engineering of capsid n sequences to transfer specific capsid n domains, surface loops or specific amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes, Shuffled or chimeric capsid proteins may also be ted by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes e.g. those encoding capsid ns of multiple different serotypes and then subsequently reassembling the fragments in a riming polymerase reaction, which may also cause crossovers in regions of ce homology. A library of hybrid AAV genes created in this way by shuffling the capsid genes of several serotypes can be screened to identify viral clones having a desired functionality. Similarly, error prone PCR may be used to ly mutate AAV capsid genes to create a diverse library of variants which may then be selected for a desired property.

The sequences of the capsid genes may also be genetically modified to introduce specific deletions, substitutions or insertions with t to the native wild-type sequence. In particular, capsid genes may be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of a capsid coding sequence, or at the N— and/or C-terminus of a capsid coding sequence.

The unrelated protein or e may advantageously be one which acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or improving the city of targeting of the vector to a particular cell population.

The unrelated protein may also be one which assists purification of the viral particle as part of the production process i.e. an epitope or affinity tag. The site of insertion will typically be ed so as not to interfere with other fiinctions of the viral particle e. g. internalisation, trafficking of the viral particle. The skilled person can identify suitable sites for insertion based on their common l knowledge. Particular sites are disclosed in Choi et a1, referenced above.

The invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome. The invention also encompasses the replacement of one or more AAV sequences or genes with ces from another virus or with chimeric genes composed ences from more than one virus. Such chimeric genes may be composed of ces from two or more related viral proteins of different viral species.

The vector of the invention takes the form of a viral vector sing the promoters and expression constructs of the invention.

The invention also provides an AAV viral particle comprising a vector ofthe invention.

The AAV les of the invention include transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype. The AAV particles of the invention also include mosaic forms wherein a mixture of unmodified capsid proteins from two or more different serotypes makes up the viral envelope. The AAV particle also es ally modified forms bearing ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting a particular cell surface receptor.

The invention additionally provides a host cell comprising a vector or AAV viral particle of the invention.

Vectors of the invention may be prepared by standard means known in the art for provision of vectors for gene therapy. Thus, well established public domain transfection, packaging and purification methods can be used to prepare a suitable vector preparation.

As sed above, a vector of the invention may comprise the full genome of a naturally occurring AAV virus in addition to a promoter of the invention or a variant thereof. However, commonly a derivatised genome will be used, for instance a derivative which has at least one inverted terminal repeat sequence (ITR), but which may lack any AAV genes such as rep or cap.

In such embodiments, in order to provide for assembly of the derivatised genome into an AAV Viral particle, additional genetic constructs providing AAV and/or helper virus ons will be provided in a host cell in combination with the derivatised genome.

These additional constructs will typically contain genes encoding structural AAV capsid proteins i.e. cap, VPl, VP2, VP3, and genes encoding other functions required for the AAV life cycle, such as rep. The selection of structural capsid proteins provided on the additional construct will ine the serotype of the packaged viral vector.

A particularly preferred packaged viral vector for use in the invention ses a derivatised genome of AAV2 in combination with AAVS or AAV8 capsid proteins.

As ned above, AAV viruses are replication incompetent and so helper virus functions, preferably adenovirus helper functions will typically also be ed on one or more additional ucts to allow for AAV replication.

All of the above additional constructs may be provided as plasmids or other al elements in the host cell, or alternatively one or more constructs may be integrated into the genome of the host cell.

Pharmaceutical Compositions, Dosages and Treatments 2016/050419 The vector ofthe ion can be formulated into pharmaceutical compositions. These compositions may comprise, in addition to the vector, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other als well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may be ined by the skilled person according to the route ofadministration, i.e. here direct retinal, subretinal or intravitreal injection.

The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally e a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution or s such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001% may be used.

For injection at the site of affliction, the active ingredient will be in the form of an aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.

Those of relevant skill in the art are well able to prepare suitable ons using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection, Hartmann's solution. vatives, stabilisers, buffers, antioxidants and/or other ves may be included, as required.

For delayed e, the vector may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in mal carrier systems according to methods known in the art.

The vectors and/or pharmaceutical compositions of the invention can be packaged into a kit.

In general, direct retinal, subretinal or intravitreal delivery of vectors of the invention, lly by injection, is preferred. Delivery to the retinal, subretinal space or intravitreal space is thus preferred. Vectors may also be introduced into rod photoreceptors in vitro followed by cell transplantation into the retina The vectors and/or pharmaceutical compositions of the invention can also be used in combination with any other y for the treatment or prevention of vision disorders.

For example, they may be used in combination with known treatments that employ VEGF antagonists, eg anti-VEGF dies such as Bevacizumab or Ranibizumab or soluble receptor antagonists such as Aflibercept, for the treatment of AMD or other eye disorders as discussed herein.

Dosages and dosage s can be determined within the normal skill of the medical practitioner responsible for administration of the composition. The dose of a vector of the invention may be determined according to various ters, especially according to the age, weight and condition of the patient to be treated, the route of administration, and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.

A typical single dose is between 1010 and 1012 genome particles, depending on the amount of retinal tissue that requires transduction. A genome le is d herein as an AAV capsid that ns a single stranded DNA molecule that can be quantified with a sequence specific method (such as real-time PCR). That dose may be provided as a single close, but may be repeated for the fellow eye or in cases where vector may not have ed the correct region of retina for whatever reason (such as surgical complication). The treatment is preferably a single permanent treatment for each eye, but repeat injections, for example in future years and/or with different AAV serotypes may be considered.

Treatments 2016/050419 Vectors of the invention may be used to treat any ocular condition in which there is dysfunction, ration or absence of cones but at least some healthy rods remain.

Cone function may be wholly or partially missing, e.g. at least 10%, at least 25%, at least, at least 50%, at least 75%, at least 80%, at least 90% or more missing. y rods are rods that are capable of performing normal or partial, e.g. at least 10%, at least %, at least, at least 50%, at least 75% or at least 90% of normal rod function in terms of perception of light at scotopic levels.

Conditions that can be treated using vectors of the ion thus include macular degeneration, achromatopsia and Leber congenital amaurosis. The macular degeneration may be age-related macular degeneration (AMD), for example wet or neovascular AMD or geographic atrophy, an inherited macular degeneration condition or an ted cone dystrophy. In some embodiments, the invention will result in the creation of a ‘pseudo-fovea’, a small patch of cone-like rods that improves vision in conditions in which foveal cones have been lost or are dysfunctional.

In l, patients to be treated with vectors of the invention will be human patients.

They may be male or female and of any age.

The ing Examples illustrate the invention.

EXAMPLES Example 1 - Methods for ArchT experiments.

Animals Wild-type mice (C57BL/6J) were purchased from Harlan Laboratories (Blackthorn, UK). CNGA3-/— and PDE6C—/— mice were bred in house. All mice were maintained under cyclic light (12 h light-dark) conditions; cage illumination was 7 foot-candles during the light cycle. All experiments were approved by the local Institutional Animal Care and Use Committees (UCL, London, UK) and conformed to the guidelines on the care and use of animals adopted by the Society for Neuroscience and the Association for Research in Vision and Ophthalmology (Rockville, MD).

Plasmid uctions, viralproduction and injection procedure The transgene construct (ArchT-EGFP) was kindly provided by Prof Ed Boyden (MIT, USA) and ns the cDNA sequence of the ArchT gene fused to the fluorescent n EGFP. The ds were packaged into AAV8 to generate recombinant AAV viral s, ho.ArchT-EGFP. Recombinant AAV8 vector was produced through a triple transient transfection method as described previously. The plasmid construct, AAV serotype-specifrc ing plasmid and helper plasmid were mixed with hylenimine (Polysciences Inc.) to form transfection complexes, which were then added to 293T cells and left for 72h. The cells were harvested, concentrated and lysed to release the vector. AAV8 was purified using AVB Sepharose columns (GE Healthcare). Both were washed in 1X PBS and concentrated to a volume of 100—150 uL. Viral particle titres were determined by comparative dot-blot DNA prepared from purified viral stocks and defined plasmid controls. Purified vector concentrations used for all experiments were 5 X 1012 viral particles/ml. Subretinal injections were performed as described previously by our group and consisted of double injections of 2ul each.

Immunohistochemistry Animals were euthanized, the eyeballs enucleated and cornea, lens and iris removed.

For retinal sections, the eyecups were fixed in 4% paraformaldehyde (PFA) for 1 hour at room temperature, before ing in optimal cutting temperature (OCT) medium. 30pm cryosections were cut in sagittal orientation, rinsed with PBS and blocked in 10% normal goat serum (NGS), 3% bovine serum albumin (BSA) and 0.1% Triton-X100.

The respective samples were incubated with primary antibodies in block on at 4°C ght using rabbit anti-cone arrestin (diluted 1:500). Following PBS washes, the respective combination of secondary antibodies (all d 1:500, life technologies) including goat anti rabbit Alexa Fluor 546 (#A1 103 5), goat anti mouse Alexa Fluor 633 (#A21052) and avidin, Alexa Fluor 633 conjugate (#S2l375) were used to label the samples before these were counterstained with DAPI and d with DAKO fluorescent mounting media (DAKO, S3023, Denmark). Images were acquired by confocal microscopy (Leica DM5500Q).

Single photoreceptor suction recordings s were dark d for 12h prior to the start of experiments. Mice were administered an overdose ofketamine-dormitor anaesthetic mix via the intra- peritoneal cavity, to induce terminal surgical-plane anaesthesia. Mice were then sacrificed by cervical dislocation and enucleated. Eyes were dissected under dim, far-red illumination. ed retinas were imbedded in 1% low melting agarose solution and then cut using a vibrotome (leica) into 23 Oum thick en face slices. Slices were mounted in a recording chamber and perfused with carbogen (95% 02 5% C02) saturated Ames medium containing 100nm 9-cis retinal (Sigma) and 0.2% BSA (Sigma). The temperature of the perfiision on was maintained at 37°C using an in-line heating element under feedback control (Scientifica). Very low resistance (1—2MQ ) patch pipettes were made from fllamented boroscilicate glass capillaries (Harvard Apparatus Ltd) using a Narishige PC-10 al puller. Pipettes were filled with external on, mounted onto the headstage and a small pressure applied across the tip (~30mbar).

Using infrared illumination and a microscope to aid visualisation the pipette was placed onto the surface ofthe retina, and then lowered ~50um into the slice, until photoreceptor segments appeared intact and neatly arranged. Slight negative pressure was applied across the pipette tip as it was advanced slowly through the retinal , using a 100ms, 10mV test pulse to monitor resistance across the pipette tip. When ance increased to ~20-30MQ light evoked responses were tested. Light stimuli from an LED light-source (peak wavelength 530nm) d to a liquid light guide, were delivered h the microscope objective (Olympus). Neutral density filters were used to precisely control the intensity of the light us. Light stimuli consisted of square wave pulses programmed using P-Clamp software ular Devices), and delivered via a DAC board (Axon Instruments) in conjunction with an LED driver (Thorlabs).

Electrophysiological ings were carried out using a Multiclamp 700B amplifier (Molecular Devices). Data was digitized at 20kHz.

MEA ings Animals were dark adapted for 12h prior to the start of experiments. Mice were administered an overdose ofketamine-dormitor anaesthetic mix via the intra— peritoneal cavity, to induce terminal surgical-plane anaesthesia. Mice were then ced by cervical dislocation and enucleated. Eyes were dissected in en (95% Oxygen, 5% Carbon Dioxide) saturated Ames medium (Sigma), under dim red light. The cornea and lens were removed, with care taken to remove as much vitreous from the surface ofthe retina as possible. The RPE was separated from the retina and a flat petal 1-3 mm across was cut away from the retinal ’cup’. This retinal petal was placed ganglion-cell side down on the surface of the lectrode array, and a circular harm constructed from unreactive platinum wire (Sigma) and nylon was used to keep the petal in position.

Throughout recordings, the tissue was perfused with carbogen saturated Ames medium (Sigma), maintained at a temperature of 36.5 degrees Celsius. For recordings that included scotopic or mesopic ions, the perfusion medium was made up to include 9-cis retinal (Sigma), at a concentration of lOOuM, in 0.2% BSA (Sigma). A perforated 60-electrode recording array, consisting of tungsten electrodes spaced 100 mm apart (Multi Channel Systems) was used to record ganglion cell extracellular ials.

Voltage changes were amplified and digitized at 50kHz by an MC Card system, using MC Rack software (MultiChannel Systems).

Electrophysiological data analysis Electrophysiological data was analysed using custom-written macros in IgorPro 6. ic currents and potentials were detected using an amplitude threshold algorithm where the threshold for event detection was set at 2 times the standard deviation of the baseline noise ally about lOpA). Detected currents and potentials were d manually through careful inspection of all electrophysiological data.

Fear conditioning Mice were trained and tested using a commercially available fear conditioning system (Med Associates). To ensure blind conditions, the experimenter performing the training and testing was always blind to the strain of mouse and treatment conditions. Briefly, the setup consisted of a conditioning chamber (20x30 cm) with a stainless steel grid floor placed inside a sound—attenuating cubicle. Mouse behaviour was monitored constantly during training and testing by means of a in infrared l video camera (30 frames/s acquisition rate) and ed illumination. Video Freeze software (Med Associates) was used to control delivery of the light stimulus and shock. The light stimulus consisted of a single LED (530 nm, Thorlabs) 5 Hz 50ms full-brightness flicker generated via an Arduino interface (Arduino Software) positioned on a side panel of the ioning chamber. To ensure that the context in which training and testing took place were different, floor and curved wall panels were inserted into the chamber for the testing session. A background white light was used to reduce chances ofrod activation and pupils were dilated with Tropicamide drops to increase the amount of light reaching the mouse retina.

Mice were placed inside the r and underwent one conditioning session, consisting of 6 pairings of a 5 5, light stimulus that co-terminated with a 2 s, 0.65 mA foot shock. Inter-trial interval was pseudo-randomized (average interval 90 3).

Following the training session mice were returned to the home cage, 24 hours after training, mice were tested for ly cued memory recall. Mice were placed in the test chamber and monitored for a total of 360 s. The conditioning light us was presented continuously for the last 120 s of the test n. All data was acquired and scored automatically by VideoFreeze software (Med ates). Briefly, the re is calibrated before placing the animal in the chamber. The software then measures the pixel changes that take place between every video frame. The motion threshold was set to be as low as possible (20 motion index units), and the continuous freezing count was set to the frame rate to ensure the most sensitive read—out of motion. To assess light cued memory recall the percentage time of freezing behaviour was averaged for the two s immediately prior to and following the light stimulus onset. Statistical significance was assessed with a one-way ANOVA Results are presented as mean :: S.E.M.

Optomotry Visual acuities were measured by observing the optomotor responses ofmice to rotating sinusoidal gratings (OptoMotry, Cerebral Mechanics). The protocol used yields independent measures of the acuities of right and left eyes based on the unequal sensitivities of the two eyes to pattern rotation as only motion in the temporal-to-nasal direction evokes the tracking se. As a result, the right and the left eyes are most sensitive to counter-clockwise (CCW) and ise (CW) rotations, respectively.

Stimuli of ent temporal frequency were used to determine the threshold at which a response was present. A double-blind two-altemative forced choice procedure was employed, in which the observer was ’ to the direction of pattern rotation, to whether it was an ArchT-treated or untreated CNGA3-/- 0r /— mouse or age- matched ype control animal (C57BL6). Visual acuity was measured in both eyes ofthe tested animal and averaged or separately analyzed for each eye after 4 trials were conducted on 4 separate days. The measurement was carried out on injected mice 3—10 weeks after treatment together with age-matched isogenic controls.

Example 2 - ArchT expression in rod photoreceptors s the ability to d with rapid non-bleaching responses Rod—mediated vision is optimized for low light levels, including single photon ion. However, rods cannot match the rapid onset and recovery of cone responses to light (Fu et al., 2007, Pugh et al., 1999). This functional difference, useful to ensure reliable vision in different environments, becomes debilitating when cone-mediated vision is lost in conditions such as in age-related macular degeneration, when the densely packed cones in the fovea degenerate (de Jong 2006). It was investigated whether if rods could respond and recover more quickly to stimuli, this would help alleviate the functional impairment caused by the loss of cones.

A fast light-driven proton pump (ArchT) (Han et a1., 2011) was expressed in rod photoreceptors. AAV8 particles carrying ArchT—EGFP under control of the Rhodopsin promoter (Rho) were injected subretinally in adult mice. Expression of chT- EGFP was limited to the membrane of rod photoreceptors (Figuresla-b). Synaptic terminals of rods expressing Rho-ArchT-EGFP and cones could be easily distinguished following immunohistochemistry e 1c). tative PCR on a sorted population of cones ed that Rho-ArchT-EGFP expression was specific to the rod population and no evident sign of toxicity was observed up to 6 months ing AAV8 injection. Expression of ArchT allowed extremely rapid light responses, while the intrinsic rod response was preserved and was comparable to that observed in non- transduced rod photoreceptors (Figure 1d). evoked currents recorded from ArchT-expressing rods trated considerably faster kinetics than the intrinsic rod currents in all mouse models tested (Figure 2a—b). These kinetics allowed light evoked currents to be modulated up to 80 Hz, far above the limits of both rods, which faltered at ~20 Hz (Figure 2a—c), and of cones (Fu et al., 2007).

Surprisingly, ArchT expression did not alter the properties of rod photoreceptors while conferring the y to respond with rapid non—bleaching responses (Figure 2e).

Example 3 - ArchT—expressing rods drive sustained RGC spiking at high light intensities and at frequencies ching those of cone photoreceptors It was next investigated whether the circuitry driven by rods would be able to follow faster-than-normal rod-driven Vision. Rod and cone pathways present some similarities and some striking differences and it is therefore not clear whether the rod circuitry can ly sustain fast ‘cone-like’ transmission (Wassle et al., 2004). Rods have been shown to contact OFF ‘cone’ bipolar cells directly (Soucy et al., 1998 and Hack et al., 1999) and paired-pulse stimulation suggests that this alternative pathway may be as fast as the cone—to-OFF-bipolar one (Li et al., 2010). However, it is not clear how sustained this response can be and whether rod (ON) bipolar cells can also sustain fast transmission. Furthermore, rod synaptic terminals have different size and ultra- structural zation ed to cones and rod bipolar cells do not contact Retinal Ganglion Cells (RGCs) directly but only through a pathway involving AII amacrine cells (Wassle et al., 2004). To investigate the maximal speed that the rod pathway can achieve, multi-electrode ings from RGCs in mouse models g cone function were performed, to isolate rod-mediated RGC output. Rod-driven ses in RGCs in non-transduced retinas were bleached at high light levels and could not follow stimulation ncies higher than ~20 Hz (Figure 2f).

On the contrary, ArchT-expressing rods drove sustained RGC spiking at high light intensities and at frequencies approaching those of cone photoreceptors (Figure 2f).

Example 4 - expression of chT-EGFP extended the sensitivity of mice lacking cone-mediated vision to bright light stimuli For this -than-normal rod vision to be , it was reasoned that mice should be able to use ArchT-mediated currents to reliably respond to bright and fast stimuli.

CNGA3'/' and PDE6C'/' mice lacking cone-mediated vision (Biel et al., 1999 and Change et al., 2009) failed to learn a fear conditioning paradigm where bright light stimuli were paired and co-terminated with a mild foot shock (Figure 3a). However, expression of Rho-ArchT-EGFP ed the sensitivity of these mice to bright light stimuli, allowing learning of the association between visual stimulus and shock (Figure 3a). Finally, it was tested whether ArchT expression red fast vision to CNGA3'/' and PDE6C'/' mice. Assessment of the speed of vision by means of Optomotor testing (Umino et all, 2008) showed that ArchT-expressing mice were able to follow stimuli faster than mice that did not o subretinal viral ions and mice that received a GFP-only vector (Figure 3b). The maximal frequency of stimuli that ArchT- expressing mice could follow was similar to that of cone photoreceptors (Figure 3b).

Together, these results show that rods can be driven faster than by their intrinsic photo- transduction cascade and that rod-driven circuits can sustain a faster signalling.

Importantly, synaptic release from rods does not require large voltage fluctuations, but small currents can instead cause sufficient voltage variations to significantly alter their synaptic transmission (Cangiano et al., 2012). This extends the use of the Invention to light levels several-fold lower than the average light levels required for optogenetic manipulation of activity in most other s (Han et al., 2011).

Example 5 - s for RPAP ments Animals C57BL6 (Harlan, UK), /- (J.R. Heckenlively, University of Michigan), Pde6c- /- (J.R. lively, University of Michigan, MI) (Chang et al., 2009), and GnatI-/- (J. Lem, Tufts University School of Medicine, MA) (Calvert et all, 2000) mice were maintained in the animal facility at University College London. Adult male and female animals were 6-12 weeks old at the time of viral injection and were used for experiments at least 2 weeks after the injection to allow for a ient expression of R9AP. All the mice used were between ages of 2 to 6 months and were age matched between groups of a given experiment. All experiments have been conducted in accordance with the Policies on the Use of Animals and Humans in Neuroscience ch and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were kept on a standard 12/12 hour light-dark cycle Plasmid constructions andproduction ofrecombinant AA V8 The murine R9ap cDNA was PCR amplified from murine retinal cDNA using primers which have been designed to encompass the whole of the coding region. The R9ap cDNA was cloned between the promoter (CMV promoter or bovine sin promoter) and the SV40 polyadenylation site. These plasmids were used to generate two pseudotyped AAV2/8 viral vectors, 8.CMV.mR9ap and rAAV2/8.Rho.mR9ap, as described below.

Recombinant AAV2/8 vector was produced through a triple transient transfection method as described previously (Gao et al., 2002). The plasmid construct, AAV serotype-specific packaging plasmid and helper plasmid were mixed with polyethylenimine to form transfection xes which was then added to 293T cells and left for 72 h. The cells were harvested, concentrated and lysed to release the vector.

The AAV2/8 was purified by affinity chromatography and concentrated using ultrafiltration columns rius Stedim Biotech, Goettingen, Germany), washed in PBS and concentrated to a volume of 100—150 ul. Viral particle titres were determined by dot-blot or by ime PCR. d vector concentrations used were 1-2 X 1012 viral particles/ml.

Electroretinogram (ERG) ERGs were recorded from both eyes after mice were dark adapted overnight using a commercially available system (Espion E2, Diagnosys LLC, , MA). The animals were anesthetized with an eritoneal injection of a 0.007 ml/g mixture of medetomidine hydrochloride (1 mg/ml), ketamine (100 mg/ml), and water at a ratio of 53:42 before recording. Pupils were fully dilated using 2.5% phenylephrine and 1.0% tropicamide. Midline subdermal ground and mouth reference electrodes were first placed, followed by positive silver electrodes that were allowed to y touch the center of the corneas under dim red illumination. A drop of Viscotears 0.2% liquid gel (Dr. Robert Winzer /OPD Laboratories, Watford, UK) was placed on top of the positive electrodes to keep the corneas moistened during recordings and the mouse was allowed to further dark-adapt for 5 minutes. Bandpass filter cutoff frequencies were 0.312 Hz and 1000 Hz. Recovery speed of photoresponse was measured using paired flash paradigm where pairs of flashes with identical saturating intensity (1.8 log.cd.s/m2) ted by various stimulus als (ISI, 05 12 4 816,32,64) 7 7 7 7 sec) were ted. In this paradigm, the 1St flash would completely suppress the electric responses of rod mechanisms which allow observation of the speed of functional recovery of the rod fiinction by presenting 2‘“d flash with different ISI.

Sufficient amount of time (150 sec) were provided between pairs of flashes to allow full recovery of the 1St flash. Then the recovery of a-wave amplitude observed should reflect the speed of deactivation of the rods in animals devoid of cone function since the flash should only bleach a fraction ) of the rhodopsin (Lyubarsky et al., 2004 and Weymouth, A.E. & Vingrys 2008). ic 6 Hz flicker intensity series were performed as usly reported with a few modifications ger et al., 2001). 17 steps of flash intensities were used ranging from -6 to 2 log.cd.s/m2 each separated by 0.5 log unit. For each step, after 10 seconds of adaptation, 600 msec sweeps were averaged 20 times using the same flash condition. Series of dark-adapted responses were also obtained using longer flashes with durations of 20, 100, and 200 msec all at 83.3 cd/mz. Standard single flash scotopic recordings were obtained from dark-adapted animals at the following increasing light intensities: -6, -5, -4, -3, -2, -1, 0, 1.0, 1.5, and 1.9 log.cd.s/m2. Photopic flash recordings were performed following 5 min light adaptation intervals on a background light intensity of 20 cd/mz, which was also used as the background light for the duration of the ings. Photopic light intensities used were -2, -1, 0, 1, 1.5, and 1.9 log.cd.s/m2.

Histologg/ SiX weeks after unilateral inal injection of rAAV2/8.Rho.mR9ap, both eyes from a Cnga3-/- mouse were y removed and snap frozen in liquid nitrogen. After cryoembedding the eye in OCT (RA Lamb, Eastborne, UK), the eyes were cut as transverse sections 15 um thick and were air-dried for 15 - 30 min. For immunohistochemistry, sections were pre-blocked in PBS containing normal donkey serum (2%), bovine serum albumin (2 %) 1 hr before being incubated with G89 antibody (1:500, Santa Cruz Biotechnology, SantaCruz, CA) for 2 hours at room temperature. After rinsing 2 x 15 min with PBS, sections were incubated with the appropriate Alexa 546-tagged secondary antibody (Invitrogen, Carlsbad, CA) for 2 hrs at room temperature (RT), rinsed and counter-stained with Hoechst 33342 - Aldrich, Gillingham, UK). Retinal sections were viewed on a confocal microscope (Leica TCS SP2, Leica Microsystems; Wetzlar, Germany).

Western blotting The eyes from a /- mouse 4 weeks after unilateral subretinal injection of rAAV2/8.Rho.mR9ap were ted. After separating the neural retina from the RPE/choroid/sclera complex, tissues were homogenized in RIPA buffer and left on ice for 20 s. The samples were centrifuged at 16,000 g for 30 minutes at 4 oC and stored in -20°C until use. Western blotting was carried out using known protocols.

Optomotor responses and contrast sensitivityfunction Contrast sensitivities and visual acuities of treated and untreated eyes were measured by observing the optomotor responses of mice to ng sinusoidal gratings (OptoMotryTM, Cerebral Mechanics, Lethbridge, AB Canada). The protocol used yields independent measures of the acuities of the right and the left eyes based on unequal ivities of the two eyes to pattern rotation: the right and the left eyes are driven primarily by counter—clockwise and clockwise rotations, respectively (Douglas et al., 2005). A mouse was placed on a small island isolated from the floor in a closed space surrounded by 4 rs with rotating sinusoidal grating with a mean illuminance of 62 cd/mz. A double-blind two-altemative forced choice procedure was employed, in which the observer was ‘blind’ to the direction of pattern rotation, to whether it was a treated or untreated Cnga3-/- mouse or age-matched wild—type control animal (C57BL6). The contrast sensitivity measured at 0.128, 0.256, 0.383, 0.511 cycles/degree presented at 6 Hz was defined as 100 divided by the lowest percent contrast yielding a threshold response. Both eyes of each mouse were tested four times on independent days. The data was projected on to a Campbell-Robson st Sensitivity Chart with sinusoidal gratings representing relative spatial frequencies, Rhodopsm measurement After fully dark-adapting the mice overnight, the mice were anaesthetized and the pupils were fully d to assess the speed of visual pigment bleaching. Then the mice were placed in a light box with a light source (7.0 mW) directly illuminating the eye for 5 minutes before the eyes were collected. In another experiment, mice were d to an identical condition as that for measuring contrast sensitivity for various durations (0, 30 60, 120 s). The mouse eyes were removed at each time point and placed in 250 pl of phosphate buffered saline and snap frozen in liquid nitrogen in a light tight tube and kept at —20 °C until use. Some eyes were collected in the dark under red nation after ght dark-adaptation of the mice. Spectrophotometric measurement of rhodopsin were performed as previously reported with minor modifications (Douglas et al., 1995). In brief, the samples were thawed at room temperature and homogenized. This and all uent operations were med under dim red nation that bleaches the visual pigments minimally. Fifty iters of n-dodecyl B-D-maltoside (200 mM; Sigma-Aldrich, St. Louis, MO) was added to every sample and the resulting mixture rotated for 2 h at room temperature, followed by 10 min centrifugation 0 g) at 4°C. The supernatant was removed and placed in a quartz cuvette in a Shimadzu UV-2101PC spectrophotometer (Shimadzu, Kyoto, . After an initial scan of the unbleached extract from 300 nm to 700 nm, the sample was exposed to monochromatic light (502 nm) for 3 minutes, shown to be enough to completely bleach rhodopsin (Longbottom et al., 2009), and rescanned. All absorption spectra were zeroed at 700 nm. Difference spectra were constructed using the pre- and post-bleach curves and the maximum optical densities at N 500 nm determined, representing the amount of the extracted visual pigment.

Example 6 - R9AP over-expression in rods and increased speed of eceptor deactivation RG89, G85, and R9AP are obligate members of the regulatory GTPase complex. To study the effect of AAV-mediated R9AP over—expression on the GTPase complex in the rods, the level and distribution of RG89 was examined following subretinal injection of rAAV2/8.Rho.mR9ap in Cnga3-/- mice. These mice have normal rod function but absent cone function and serve as a model of achromatopsia. Four weeks later, treated retina showed sed immunoreactivity against RG89 throughout the eceptor layer in the treated compared to the untreated retinas (Figure 4A).

Westernblot is further confirmed the increased RG89 n expression in the treated retina (Figure 4B). These results indicated that the over—expression of R9AP using AAV2/8 effectively increased the level of catalytic ent RG89 and the GTPase complex.

Next the fiJnctional effect of AAV2/8-mediated R9AP over-expression on rod phototransduction was studied by applying paired-flash ERG rsky and Pugh 1996). In this paradigm, a pair of identical flash intensity is delivered with a variable inter-stimulus interval and the recovery of the second response relative to the first is WO 35457 measured. In the rod photoreceptor pathway, the speed of the a-wave (originating from photoreceptors) recovery is dependent on the speed of the deactivation, It was found that the time nt (6) for 50% recovery of a—wave amplitude was d by ~60% in the Cnga3-/— eyes injected with rAAV2/8.CMV.mR9ap (o = ~ 2.99 sec) ed to the ted eyes (6 = ~ 7.38 sec; Figure 4C). rly, an increased speed of a- wave recovery was observed using rhodopsin promoter (rAAV2/8.Rho.mR9ap, c5 = ~ 2.74 sec, Figure 4C) in the same mouse line (Cnga3-/-) or the same virus (rAAV2/8.CMV.mR9ap) in another efective mouse line -/—; Figure 8).

These observations indicated that the subretinal injection of rRAAV2/8.CMV.mR9ap or rAAV2/8.Rho.mR9ap can significantly increase the deactivation speed of the rod phototransduction through sing the level of RGS9 and GTPase complex.

Example 7 - “Photopic shift” of rod function by over-expression of R9AP To investigate if an increased deactivation speed achieved by overexpression of R9AP and GTPase complex in the rods could alter the operating range of the photoreceptor function, dark-adapted 6 Hz flicker ERGs were recorded using incremental flash intensities. The eyes treated with rAAV2/8.CMV.mR9ap or rAAV2/8.Rho.mR9ap showed increased responses to brighter flashes compared to the untreated eyes. This resulted in an elevation the upper threshold ofthe response by up to ~2 log units (Figure 5A), with little effect on the maximal photoresponse (151 i 17 uV in the treated vs 162 :: 29 uV in the untreated eyes; average i standard error of the mean). As expected, this “photopic shift” in the operating range of the rods was accompanied by a reciprocal elevation the lower threshold of the response by up to ~15 log units. Meanwhile, upper threshold ofERG responses of wild-type eyes and GnatI-/- eyes, both with functional cones, were elevated by ~40 log units compared to that of the untreated Cnga3-/— eyes.

Similar results were obtained when rAAV2/8.Rho.mR9ap was injected into Pde6c-/- mice (Figure 9). As a consequence, the treatment allowed the rods to d to flashes of longer durations (Figure 5B) and to flashes under a solating background WO 35457 illumination e 5C). These include conditions where the untreated rods showed virtually no response.

Taken together, these results ished that R9AP-over expression in rods results in their desensitization and endows the cells to gain photopic filnction in exchange for scotopic function. This therapeutic induction of “photopic shift” of the rod function lasted at least for 5 months without overt evidence of retinal degeneration (Figure 10).

Meanwhile, the treatment of wildtype mice using the same viral vectors failed to show a measurable change in l function (Figure 11).

Example 8 - Rod bipolar pathway accommodates the transmission of altered rod function.

This work has established that an pression of R9AP in rods s in faster photoreceptor deactivation cs and allows the neuron to respond to larger amount of photons, Meanwhile, the rated deactivation should also result in a shorter duration of the neurotransmitter release at the photoreceptor synaptic terminal.

Therefore, it was assessed whether the down-stream rod bipolar signaling is affected by the treatment. First the speed and the extent of transmission of signals from the photoreceptors to the r cells to a short single flash was studied by measuring the implicit time and amplitudes of the a-wave (originating from the photoreceptors) and b-wave (originating from the r cells) using ERG (Figure 6a). Overall, slightly smaller but a nearly identical intensity-response curve was observed for the treated and the untreated eyes for both a—wave and b-wave. The small difference observed may reflect either the true consequence of rated photoreceptor deactivation or merely the neural damage induced by the subretinal injection. The a-wave implicit time marks the point at which the bipolar cell-driven b-wave becomes detectable. We also observed a small delays in the a-wave and the b—wave implicit times, indicating a modest delay exists in the transmission of neural signals from eceptors to bipolar cells.

Nevertheless, a relatively large variation of ERG responses between individuals indicate a small delay or ion in rod se do not necessarily translate into Visual dysfunction (Birch and Anderson 1992).

Therefore, these results indicated that, in principle, the r cells almost fiJlly accommodate the alteration of photoreceptor on and, importantly, display an appropriate dose-response onship.

Example 9 - R9ap over-expression results in improved Contrast Sensitivity Function Next, it was asked if the “photopic shift” of the rod function by R9AP over-expression were consequently translated into improved Visual performance under light by measuring optokinetic response to rotating sinusoidal gratings under the brightest recording condition possible with a standard computer monitor (62 cd/mz) (Carvalho et al., 2011). The unique advantage of this oral test is that Visual function of each eye can be studied separately; the function of the right eye can be probed by responses to counterclockwise (CCW) gratings and the left eye by clockwise (CW) stimuli (Douglas et al., 2005). The spatial contrast sensitivity function (CSF) was studied with a fixed temporal frequency of 6.0 Hz and found that Cnga3-/— mice had reduced CSF ed to the wildtype mice e 7). CSF, a function of contrast sensitivity and Visual acuity, displayed the estimate range of Visual perception (animals could presumably perceive the gratings under the curve but not above, Figure 7A).

Intriguingly, an 80-fold (P = 0.005) and 54-fold (P = 0011) increase in the sensitivity using gs of both 0.128 and 0.256 cycles/degree (c/d), respectively, was observed when contrast sensitivity of the treated and the ted eyes were compared (Figure 7A left panel). No clear alteration of contrast sensitivity was observed for gratings of 0.383 (P = 0.056) and 0.511 (P = 0.111) c/d. Interestingly, the average sensitivity of the treated eye in /— mice exceeded that of the wild-type controls with normal cone function. However, when wild-type mice were treated with the same Viral uct, CSFs were not different between the d and the untreated eyes (Figure 12).

Having established that R9AP overexpression results in gain of visual performance when g maximally bright monitor settings, it was sought to determine ifthis gain- of—vision is sustainable. This is of a valid concern considering that the ration of visual pigment in rods is known to be considerably slower than that of the cones (Wang and Kefalov 2011). First, it was assessed if the treatment results in tion in the speed of visual pigment ing. It was found that an exposure of the treated and untreated eyes to a bright light for 5 minutes did not yield any difference in the levels of residual bleachable visual pigment (Figure 7B lower left panel). Second, the amount of bleachable sin in the eye was studied after exposing the Cnga3-/- mice for a variable amount of time to the same experimental condition carried out for CSF measurement. The results showed that visual t level remained stable without evidence of reduction throughout 2 hours’ exposure to the visual stimuli similarly for the treated and the untreated eyes (Figure 7B lower right panel). These results indicated that the gain of visual perception in the treated Cnga3-/— mice is supported by sufficient supply of rhodopsin molecules and is sustainable.

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Claims (14)

1. Use of an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a gene product that is sensitive and/or that modulates nous light-sensitive 5 signalling in a photoreceptor cell, in the manufacture of a medicament for improving vision in a patient with cone photoreceptor dysfunction and/or degeneration by introduction of said nucleic acid into healthy rod photoreceptors in the retina of the patient and expression of said gene product therein, such that the range of light intensities to which the rod photoreceptor responds is extended and/or the speed at which the rod photoreceptor 10 ds to light is increased, wherein the gene product is ArchT or R9AP; and the capsid of the AAV vector is derived from AAV8. 15

2. Use of an AAV vector according to claim 1, wherein the genome of the AAV vector is d from AAV2.

3. Use of an AAV vector according to claim 1 or 2, wherein the patient suffers from macular degeneration, achromatopsia or Leber congenital amaurosis.

4. Use of an AAV vector according to claim 3, wherein the macular degeneration is age-related macular degeneration (AMD), an inherited macular degeneration condition or an inherited cone dystrophy. 25

5. Use of an AAV vector according to claim 4, wherein the AMD is wet or neovascular AMD or geographic atrophy.

6. Use of an AAV vector according to any one of the ing claims, wherein rod eceptor signalling is extended into the mesopic and/or photopic illumination range.

7. Use of an AAV vector according to any one of the ing claims, n the rods exhibit improved modulation strength and/or faster activation/inactivation kinetics.

8. Use of an AAV vector according to any one of the preceding , wherein the medicament is intended to be introduced into rod photoreceptors in vitro and transplanted into the retina.

9. Use of an AAV vector according to any one of the preceding claims, wherein the mesopic and/or photopic vision of the patient is improved.

10. Use of an AAV vector according to any one of the preceding claims, wherein the 10 nucleic acid is intended to be expressed under the control of a photoreceptor-specific or photoreceptor-preferred promoter.

11. Use of an AAV vector according to claim 10, wherein said photoreceptor-specific or photoreceptor-preferred promoter is a rod-specific or rod-preferred promoter.

12. Use of an AAV vector according to any one of claims 1 to 11, wherein the nucleic acid is intended to be expressed under the control of a rod-specific promoter.

13. Use of an AAV vector according to any one of claims 10 to 12, wherein the nucleic 20 acid is intended to be expressed under the l of a sin (Rho), Neural retinaspecific leucine zipper protein (NRL) or Phosphodiesterase 6B (PDE6B) promoter.

14. Use of an AAV vector ing to any one of claims 1 to 13 wherein the medicament is intended to be used in ation with another therapy.