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Gene therapy treatment of color blindness in adult primates

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We have used gene therapy in which an adeno-associated viral vector containing a human photopigment gene was injected in the eyes of adult dichromatic squirrel monkeys, with the intent of adding red-green color vision. The goal was to exploit the capricious nature of viral infection to transduce only a subset of cones, producing a retinal region with two randomly-interspersed cone types absorbing in the middle-to-long wavelengths. To test color vision, we adapted the Cambridge Colour Test for use with animals. The monkeys' pre-therapy behavior was highly reliable, and they always failed to make “red-green” color discriminations as predicted from their known cone complement. After treatment, the monkeys showed marked improvement in red-green color vision. Conventional wisdom has held that “critical periods” exist for the development of new visual capacities. This has raised the concern that treating adults with congenital conditions could be impossible. To the contrary, here, adult monkeys that had been red-green colorblind from birth obtained a new dimension of color vision as the result of adding a third photopigment. The successful treatment of adult primates with a congenital color vision deficiency is encouraging for the possibility of gene therapy to treat a variety of inherited vision disorders in humans.
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LETTERS
Gene therapy for red
green colour blindness in adult
primates
Katherine Mancuso
1
, William W. Hauswirth
2
, Qiuhong Li
2
, Thomas B. Connor
3
, James A. Kuchenbecker
1
,
Matthew C. Mauck
3
, Jay Neitz
1
& Maureen Neitz
1
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 trichro-
matic colour vision. This opened a new avenue to explore the
requirements for establishing the neural circuits for a new dimen-
sion 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 colour-
deficient primates was sufficient to produce trichromatic colour
vision behaviour. Thus, trichromacy can arise from a single addi-
tion of a third cone class and it does not require an early develop-
mental 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 blind
2
. 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 rods
3
. To provide the receptoral basis for trichromacy,
animals received three 100-ml injections (containing a total of
2.7 3 10
13
viral particles) in each eye, which produced a relatively
uniform, third submosaic of approximately 15–36% of M cones that
coexpressed the transgene (Fig. 1e, f).
Before treatment, monkeys were trained to perform a computer-
based colour vision test, the Cambridge Colour Test
4,5
, which was
modified for use with animals
6
(Fig. 2a). 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 blue-
green 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 of 2499 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
1
Department of Ophthalmology, Box 356485, University of Washington, 1959 North East Pacific Street, Seattle, Washington 98195, USA.
2
Department of Ophthalmology and Powell
Gene Therapy Center, University of Florida, 1600 South West Archer Road, Gainesville, Florida 32610, USA.
3
Department of Ophthalmology, Medical College of Wisconsin, 925 North
87th Street, Milwaukee, Wisconsin 53226, USA.
28.3
1.35
f
15 deg
e
1 mm
40.0 μm
25 μm
d
1 mm
c
51.7
nV deg
–2
nV deg
–2
2.01
15 deg
b
a
CHOPS 2053
TR LCR PP
SD/
SA
RHLOPS PA
1
TR
5 3
Not I Not I
0.5 kb
Figure 1
|
rAAV2/5 vector produced functional L-opsin in primate retina.
a
, Molecular map. LCR, locus control region; PA
1
, polyadenylation signal;
PP, proximal promoter; RHLOPS, recombinant human L-opsin cDNA; SD/
SA, splice donor/acceptor; TR, terminal repeats.
b, Red light mf-ERG
stimulus.
c, mf-ERG 40 weeks after two injections (yellow circles) of a
mixture of L-opsin- and GFP-coding viruses. Grey lines show borders of
highest response. For comparison, the inset shows mf-ERG 16 weeks after
injection; there was no reliable signal from L-opsin, unchanged from
baseline. High responses in far peripheral retina were measured reliably and
may have originated from offshoot of one of the injections.
d, Fluorescence
photographs from a similar retinal area as in
c; grey lines from c were copied
in
d. e, Confocal microscopy showed a mosaic pattern of GFP expression in
5–12% of cones. Because GFP-coding virus was diluted to one-third
compared to L-opsin virus, an estimated 15–36% of cones in behaviourally
tested animals express L-opsin.
f, Mf-ERG from a behaviourally tested
animal 70 weeks after three injections of L-opsin virus.
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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 490 nm 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 endo-
genous 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 weeks
8
. 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 (Fig. 1d).
The two dichromatic monkeys who participated in behavioural
tests of colour vision were treated with L-opsin-coding virus only.
Although the elongated pattern producedby two injections in Fig.1c, d
allowed mf-ERG validation, the treatment goal was to produce a
homogeneous region, as resulted from three injections shown in
Fig. 1f, 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 ‘M 1 L cone’ submo-
saic was absent, they would remain dichromatic—the hallmark of
which is having two hues that are indistinguishable from grey
(Fig. 2d). 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 495 nm. At the limit, an increase in
spectral sensitivity would shift the monkeys’ neutral point towards that
of individuals with only S and L cones, near the dominant wavelength
of 505 nm (Fig. 2d, dashed blue lines). 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 to Fig. 2e,
obtained from a trichromatic female control monkey.
Daily testing continued after treatment. After about 20 weeks post-
injection (Fig. 3a, arrow), the trained monkeys’ thresholds for blue-
green and red-violet (dominant wavelengths of 490 and 2499 nm,
respectively; Fig. 3b, c) 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 monkey
8
.
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 domi-
nant wavelengths of 490 nm (Fig. 3a, triangle) and 2499 nm, indi-
cating 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 dupli-
cate messenger RNA start site that may have interfered with expres-
sion (see Methods). Three monkeys received injections of this vector,
containing an average of 1.7 3 10
12
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 experi-
ments reported here, we removed the extra 146-bp segment and also
increased the amount of viral particles delivered per eye by approxi-
mately 16-fold, to 2.7 3 10
13
. 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, individuals with L but no M cones (termed deuteranopes)
have a relatively enhanced sensitivity to red light, but they are still as
0.16
–559 465 485 490 535
Dominant wavelength (nm)
Dominant wavelength (nm)
567 583 –499
Thresholds (vector length)Thresholds (vector length)
Thresholds (vector length)
0.12
0.08
0.04
0.24
0.20
0.16
0.12
0.08
0.04
0.00
0.24
0.20
0.16
0.12
0.08
0.04
0.00
–541 –567 478 485 507 554 576 595
Dominant wavelength (nm)
–541 –567 478 485 507 554 576 595
0.00
0.16
–559 465 485 490 535
Dominant wavelength (nm)
567 583 –499
Thresholds (vector length)
0.12
0.08
0.04
0.00
a
bc
de
Figure 2
|
Pre-therapy colour vision and possible treatment outcomes.
a
, Colour-vision stimuli examples. b, Pre-therapy results, monkey 1. Hues
tested are represented as dominant wavelengths rather than u9, v9
coordinates. If a hue could not be reliably distinguished at even the highest
saturation, the extrapolated threshold approached infinity.
c, Pre-therapy
results, monkey 2.
d, e, Possible experimental outcomes: monkeys could
have a relative increase in long-wavelength sensitivity, but remain
dichromatic (dashed lines,
d); theoretical colour spectrum appearances for a
dichromat and a possible ‘spectral shift’ are shown. Alternatively,
dichromatic monkeys could become trichromatic. Results from a
trichromatic female control monkey are plotted (dashed line,
e). Error bars
denote s.e.m.; n varied from 7–11.
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dichromatic as individuals with M but 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 (see
Fig. 2d), 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 inter-
mediate dichromatic forms that could arise from expressing combina-
tions of L and M pigments. As shown in Fig. 3b, c, both monkeys’
measured thresholds for these extra hues were similar to their thresh-
olds 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 red-
violet (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 treated monkeys’
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 loss
1
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 trichro-
macy would require evolutionary/developmental changes, in addi-
tion to acquiring a third cone type. For example, L- and M-opsin-
specific genetic regulatory elements might have been required to
direct the opsins into distinct cone types
9
that would be recognized
by L- and M-cone-specific retinal circuitry
10
, and to account for
cortical processing, multi-stage circuitry
11
might have evolved spe-
cifically 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 mice
12
; however, the point has
been made that such plasticity need not indicate that any rewiring of
the neural circuitry has occurred
13
. 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 adult monkeys 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 know whether the animals experi-
ence new internal sensations of red and green. Nonetheless, we do
know that evolution acts on behaviour, not on internalized experi-
ences, 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-exist-
ing 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 amaur-
osis
14–16
. Thus far, treatment has been administered to individuals
who have suffered retinal degeneration from the disease. The experi-
ments reported here are, to our knowledge, the first to use gene
therapy in primates to address a vision disorder in which all photo-
receptors 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.
METHODS SUMMAR Y
Confocal microscopy. The animal in Fig. 1c, d succumbed to respiratory illness,
unrelated to gene therapy, approximately 2 years and 3 months after injection.
100
0.24
0.20
0.16
0.12
0.08
0.04
0.00
0.24
0.20
0.16
0.12
0.08
0.04
0.00
Threshold (log vector length)Threshold (vector length)Threshold (vector length)
10
1
0.1
P1 P2 P3 0 5
–541
–559
–567
465
478
481
485
490
496
500
507
535
554
567
576
583
595
–499
11 13 15 17 19 22 24 26 28 30 32 79 81
0.01
Squirrel monkey 1 threshold comparison over time
Squirrel monkey 1 pre- and post-injection thresholds
Squirrel monkey 2 pre- and post-injection thresholds
Time since injection (weeks)
Dominant wavelength (nm)
–541
–559
–567
465
478
481
485
490
496
507
535
554
567
576
583
595
–499
Dominant wavelength (nm)
a
b
c
Figure 3
|
Gene therapy produced trichromatic colour vision. a, Time
course of thresholds for the blue-green confusion colour, dominant
wavelength of 490 nm (circles), and a yellowish colour, dominant
wavelength of 554 nm (squares). A logarithmic scale was used to fit high
thresholds for the dominant wavelength of 490 nm; significant improvement
occurred after 20 weeks. Enclosed data points denote untreated dichromatic
monkey thresholds, dominant wavelengths of 490 nm (triangle) and 554 nm
(diamond).
b, c, Comparison of pre-therapy (open circles, solid line) and
post-therapy (solid dots, dashed line) thresholds. Enclosed data points are
dominant wavelength 490 nm thresholds when tested against a red-violet
background (dominant wavelength of 2499 nm); pink
triangles show trichromatic female control thresholds. Error bars represent
s.e.m.; n varied from 7–11.
LETTERS NATURE
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The retina was fixed in 4% paraformaldehyde in PBS, and rinsed in PBS with
10% and 30% sucrose. It was sequentially incubated with 10% normal donkey
serum, rabbit monoclonal antibody to M/L-opsin (Chemicon, AB5405), and a
Cy3 (red)-conjugated donkey anti-rabbit antibody (Jackson Immunoresearch).
Confocal images were analysed using ImageJ (http://rsbweb.nih.gov). In the
middle panel of Fig. 1e, magenta dots mark cone locations, and the red anti-
M/L-opsin antibody staining was removed to show GFP-expressing (green) cells
more clearly.
Behavioural colour vision assessment. A three-alternative forced-choice model
in which position and saturation of the stimulus was randomized between trials
was used. Monkeys had to discriminate the location of a coloured patch of dots
that varied in size and brightness, surrounded by similarly varying grey dots.
When animals touched the coloured target, a positive tone sounded and a juice
reward was given; the next stimulus appeared immediately. (The squirrel
monkey shown in Fig. 2c is drinking a reward from a previous trial). If the wrong
position was chosen, a negative tone sounded, and a 2–3-s ‘penalty time’
occurred before the next trial.
For each hue, monkeys were tested on up to 11 different saturations ranging
from 0.01 to 0.11 in u9, v9 colour space (CIE 1976) and a threshold was calculated,
which was taken as the saturation required to reach a criterion of 57% correct, the
value determined to be significantly greater than chance (33% correct, P 5 0.05);
see ref. 6 for full details. All procedures were conducted in accordance with the
guidelines of the US National Institutes of Health about the care and use of
animals.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 19 June; accepted 14 August 2009.
Published online 16 September 2009.
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Acknowledgements This work was supported by the National Institutes of Health
grants R01EY016861 (M.N.) and R01EY11123 (W.W.H.); Research Training
Program in Vision Science Grant T32EY014537; NEI Core Grants for Vision
Research P30EY01931, P30EY01730 and P30EY08571; the Harry J. Heeb
Foundation, the Posner Foundation, the Macular Vision Research Foundation, the
Foundation Fighting Blindness, Hope for Vision, and Research to Prevent Blindness.
We would like to thank V. Chiodo, S. Boye, D. Conklyn, P. M. Summerfelt,
K. Chmielewski and K. L. Gunther for technical assistance. J.N. is the Bishop
Professor in Ophthalmology, M.N. is the Ray Hill Professor in Ophthalmology, and
W.W.H. is Rybaczki-Bullard Professor of Ophthalmology.
Author Contributions Experiments and data analysis were performed by K.M.,
T.B.C., J.A.K., M.C.M., J.N. and M.N. Cone-specific expression of the gene therapy
vector was developed and validated by Q.L., and W.W.H. constructed the vector
and packaged it into adeno-associated virus and provided dosage guidance. All
authors contributed to data interpretation. The manuscript was written by K.M.,
J.N. and M.N. and incorporates comments by all others.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare competing financial interests:
details accompany the full-text HTML version of the paper at www.nature.com/
nature. Correspondence and requests for materials should be addressed to J.N.
(jneitz@uw.edu).
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METHODS
Viral vector. CHOPS2053 was a 2.1-kilobase (kb) fragment containing the locus
control region and proximal promoter upstream of the human X-chromosome
opsin gene array
9,17
. These elements (also known as pR2.1) have been shown to
target transgene expression to mammalian L/M cones
3,18
. RHLOPS was a 1.2-kb
fragment containing recombinant human L-opsin cDNA. A clone of the human
L-opsin cDNA
19
, known as hs7, was generously provided by J. Nathans. The
QuickChange kit (Stratagene) was used to convert codon 180 so that it would
encode a human L pigment maximally sensitive to 562 nm
20
. The virus was made
using the genome from rAAV serotype 2 and the capsid from serotype 5, and the
preparation had 9 3 10
13
DNase-resistant vector genome containing particles
per ml. To prevent vector aggregation, 0.014% Tween-20 was added to the final
vector preparation. A total of 2.7 3 10
13
viral particles were injected per eye.
An earlier version of the L-opsin-coding rAAV2/5 used in previous un-
successful experiments contained an extra 146-bp segment between the splice
donor/acceptor site and the translational start codon of the L-opsin gene that
had been carried over from the cloning vector. Because we were concerned that
this fragment may have interfered with transgene expression, a second version of
L-opsin rAAV2/5 in which the extra 146 bp had been removed was used in later
experiments described here. In addition to modifying the vector, we also
increased the amount of viral particles delivered per eye by approximately 16-
fold, from 1.7 3 10
12
to 2.7 3 10
13
. Thus, we cannot conclude from this set of
experiments what exact titre of viral particles was required to produce the effects
on colour vision behaviour, or exactly what effects, if any, the extra 146 bp had on
transgene expression in earlier unsuccessful attempts.
The single-stranded DNA genome of conventional rAAV vectors, including
rAAV2/5 used here, is devoid of Rep coding sequences. Thus, the vector genome
is stabilized predominantly in an episomal form; however, the potential for
integration exists
21
. According to NIH guidelines, the viral vector used here is
rated biosafety level 1 (BSL1), and animal biosafety level 1(ABSL1) meaning that
no special precautions were required in handling the virus or animals treated
with the virus. After treatment, squirrel monkeys had an increase in AAV anti-
body titres, ranging from 4–12-fold. Antibody titres remained unchanged in
untreated control animals who were housed with treated animals.
Subretinal injections. Subretinal injections were performed by a vitreo-retinal
surgeon (T.B.C.) using a KDS model 210 syringe pump under a stereomicro-
scope. A 500-ml Hamilton Gastight (1750TTL) Luer Lock syringe was connected
to 88.9 cm of 30 gauge teflon tubing with male Luer Lock adapters at both ends
(Hamilton 30TF double hub), which was then connected to a 30-gauge Becton
Dickinson Yale regular bevel cannula (ref 511258) that was manually bent to
produce a 135u angle 1.5-mm from the tip. All components were sterilized before
use. The syringe and tubing were filled with sterile lactated Ringers solution to
produce a dead volume of approximately 210 ml. Just before injection, 300 mlof
rAAV was withdrawn using a rate of 100 ml min
21
.
Squirrel monkeys were anaesthetized using intramuscular injectionsof ketamine
(15 mg kg
21
) and xylazine (2 mg kg
21
); atropine (0.05 mg kg
21
) was also given to
reduce airway secretions. The eye was dilated with 2–3 drops of tropicamide (1%)
and treated with one drop each of betadine (5%), vigamox (0.5%) and propara-
caine (1%). Subconjunctival injection of 0.1 ml lidocaine (2%) was given, and the
anterior portion of the eye was exposed by performing a temporal canthotomy
followed by limited conjuntival peritomy. Eyelids were held open with a speculum
designed for premature infants. A temporal sclerotomy was made 1-mm posterior
to the limbus with a 27-gauge needle, through which the injection cannula was
inserted. Three subsequent 100-ml injections were made at different subretinal
locations using an infusion rate of 1,060 mlmin
21
. Post-procedure, 0.05 ml each
of decadron (10 mg ml
21
), kenalog (40 mg ml
21
) and cephazolin (100 mg ml
21
)
were injected subconjunctivaly; one drop each of betadine (5%) and vigamox
(0.5%) and a 0.6-cm strip of tobradex (0.3% tobramycin, 0.1% dexamethasone)
ointment were applied topically; 10–20ml of subcutaneous fluids (sterile
lactated Ringers) was also given. Subsequent administration of steroids and
analgesics was administered as needed post-procedure for potential inflammation
or discomfort.
17. Wang, Y. et al. A locus control region adjacent to the human red and green visual
pigment genes. Neuron 9, 429
440 (1992).
18. Mauck, M. C. et al. Longitudinal evaluation of expression of virally delivered
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282 (2008).
19. Nathans, J., Thomas, D. & Hogness, D. S. Molecular genetics of human color
vision: the genes encoding blue, green, and red pigments. Science 232, 193
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20. Neitz, M., Neitz, J. & Jacobs, G. H. Spectral tuning of pigments underlying red-
green color vision. Science 252, 971
974 (1991).
21. Bu¨ning, H., Perabo, L., Coutelle, O., Quadt-Humme, S. & Hallek, M. Recent
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... 36 These results suggest an ongoing process of gene conversion for some human photopigment opsin genes, and further work will provide a more complete understanding of its dynamics and what specific opsin gene features the homogenizing conversion is acting on. 37 Go Back To Main Article. ...
... suggests that one color vision test widely used in industry and the military can existence of specialized groups of color observers in a population would create problems for a population's evolution of a shared color naming and categorization system. That is, if subsets of a society's individuals use different perceptual categories for identifying objects, how can successful communication occur among all members, and how could a shared color communication system evolve?We used computer simulation approaches from evolutionary game theory to investigate such questions using simulated color category learning scenarios.37,38,39,40 Our results showed no obstacles to evolving stable categorization solutions in populations that include agents modeled with normal, deficient and putative tetrachromat discrimination data. ...
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