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Effective gene therapy for an inherited CNS disease in a large animal model.

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Effective Gene Therapy for an Inherited
CNS Disease in a Large Animal Model
Charles H. Vite, DVM, PhD,1,2 Joseph C. McGowan, PhD,3 Sumit N. Niogi, BS,1 Marco A. Passini, PhD,1,2
Kenneth J. Drobatz, DVM, MSCE1 Mark E. Haskins, VMD, PhD,1 and John H. Wolfe, VMD, PhD1,2
Genetic diseases affecting the brain typically have widespread lesions that require global correction. Lysosomal storage
diseases are good candidates for central nervous system gene therapy, because active enzyme from genetically corrected
cells can be secreted and taken up by surrounding diseased cells, and only small amounts of enzyme (<5% of normal)
are required to reverse storage lesions. Injection of gene transfer vectors into multiple sites in the mouse brain has been
shown to mediate widespread reversal of storage lesions in several disease models. To study a brain closer in size to the
human brain, we evaluated the extent of storage correction mediated by a limited number of adeno-associated virus
vector injections in the cat model of human ␣-mannosidosis. The treated cats showed remarkable improvements in
clinical neurological signs and in brain myelination assessed by quantitative magnetic resonance imaging. Postmortem
examination showed that storage lesions were greatly reduced throughout the brain, even though gene transfer was
limited to the areas surrounding the injection tracks. The data demonstrate that widespread improvement of neuropathology in a large mammalian brain can be achieved using multiple injection sites during one operation and suggest that
this could be an effective treatment for the central nervous system component of human lysosomal enzyme deficiencies.
Ann Neurol 2005;57:355–364
A large number of single-gene disorders affect the central nervous system (CNS).1 Many of these diseases are
caused by deficiencies in metabolic pathways, which affect cells throughout the brain. Somatic gene transfer
has the potential to permanently correct the underlying
metabolic deficiency by transferring a normal copy of a
defective gene into a patient’s own cells. However,
gene transfer is generally restricted to areas near injection sites. Although global correction of genetic metabolic disease by gene therapy can be achieved under
certain circumstances in the 400mg mouse brain,2,3 it
would be necessary to achieve a scale more than 1,000fold greater to treat the human infant brain. We hypothesized that by developing effective methods in the
30g cat brain, we would show the potential for therapeutic efficacy likely to be encountered in the human
The cat model offers a number of advantages for investigating translational approaches to somatic gene
therapy of the CNS. First, the feline nervous system
has been well characterized functionally, anatomically,
and physiologically, and the physical organization of
the cat brain is more similar to the human brain than
is the murine brain.4,5 Second, many naturally occurring inherited metabolic disorders affect the cat
brain.2,3,6,7 Third, the background genetic heterogeneity among relatively noninbred cats is similar to the
genetic diversity of affected human populations, which
is unlike the relative genetic homogeneity of mouse
models. Fourth, adequate numbers of affected individuals and healthy siblings can be produced for matched
treatment and control groups to achieve statistical
power.8 –12 Fifth, because of improved resolution of
brain structures, the large size of the cat brain permits
the study of regional correction of neuropathology using noninvasive imaging methods.13–16
We evaluated gene therapy in brains of cats affected
with ␣-mannosidosis (AMD), a lysosomal storage disease caused by a deficiency of lysosomal ␣-mannosidase
(LAMAN) activity. Human AMD is characterized by
predominantly progressive neurological dysfunction
and skeletal abnormalities.17–20 The disease in the cat
is caused by a 4bp deletion in the feline ␣-mannosidase
gene (fMANB) and is characterized by clinical signs,
neuropathology, and biochemical abnormalities similar
to those seen in human patients.9,21–29 AMD cats were
From the 1School of Veterinary Medicine, University of Pennsylvania; 2Children’s Hospital of Philadelphia, Philadelphia, PA; and
US Naval Academy, Annapolis, MD.
This article includes supplementary materials available via the Internet
Received Aug 24, 2004, and in revised form Nov 30. Accepted for
publication Dec 1, 2004.
Current address for S. Niogi: School of Medicine, Cornell University, New York, NY 10021. Current address for Dr Passini: Genzyme Corporation, Framingham, MA 01701.
Published online Feb 24, 2005, in Wiley InterScience
( DOI: 10.1002/ana.20392
Address correspondence to Dr Wolfe, 503 Abramson Research Center, Children’s Hospital of Philadelphia, 3516 Civic Center Blvd.,
Philadelphia, PA 19104-4318. E-mail:
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
injected intracerebrally at multiple sites with an adenoassociated virus (AAV) packaged in a serotype 1 vector
(AAV1) expressing a wild-type copy of fMANB complementary DNA (cDNA). The treated cats expressed
LAMAN activity throughout the brain, showed widespread correction of neuropathology, and demonstrated
significantly diminished effects of the clinical neurological syndrome.
Materials and Methods
Cats were raised under National Institutes of Health and US
Department of Agriculture guidelines, and all studies were
approved by the Institutional Animal Care and Use Committee. Affected cats were bred from carrier cats, and all were
genotyped for the mutant allele.21 Physical and neurological
examinations were performed weekly from birth up until the
animals were killed.
Vector Production
The AAV vector genome contained AAV2 inverted terminal
repeats (ITRs) flanking a transcription unit containing
fMANB under the control of a cytomegalovirus promoter
and bovine growth hormone poly A sequences. The vector
plasmid was packaged into an AAV1 capsid, which had a
titer of 6.4 ⫻ 1012 genome equivalents per milliliter.
Intraparenchymal Injection of Viral Vectors
At 8 weeks old, six cats homozygous for the mutant allele
were anesthetized with intravenous propofol (up to 6mg/kg;
Abbott Laboratories, Chicago, IL), endotracheally intubated,
maintained under anesthesia with isoflurane (IsoVet;
Schering-Plough, Omaha, NE), and placed in a nonferrous
stereotaxic head holder designed by the investigators. A midline incision was made over the dorsum of the calvaria, and
holes (1mm in diameter) were drilled in each side of the
calvaria over the brain regions of interest, which included the
cortex of the frontal lobe, the head of the caudate nucleus,
the thalamus, the corona radiata of the frontal lobe, the centrum semiovale, the internal capsule, and the cerebellum. A
25␮l Hamilton syringe with a 26-gauge needle was used to
administer the AAV1-fMANB vector. The needle was placed
into each drill hole and lowered to the base of the brain; 2␮l
vector was injected, and 2 minutes was allowed for vector
diffusion. The needle was withdrawn vertically at 0.25cm
distances, and the injection procedure was repeated until the
needle was no longer in the brain. A total of 125␮l vector
was administered per cerebral hemisphere of each cat. A total
of 25␮l vector was administered per cerebellar hemisphere.
In all cats, both cerebral hemispheres and both cerebellar
hemispheres received the AAV1-fMANB vector.
Magnetic Resonance Imaging
Magnetic resonance imaging (MRI) was performed on anesthetized cats (normal: n ⫽ 3; AMD: n ⫽ 3; treated: n ⫽ 3).
Cats were imaged at 16 weeks old using a dedicated research
1.5Tesla MRI scanner (Signa; General Electric Corporation,
Milwaukee, WI). The imaging session consisted of a sagittal
spin-echo short repetition time (TR), short TE localizer, ax-
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ial fast spin-echo imaging with long TR/TE, and magnetization transfer imaging using previously described methods.9
Data were transferred to a Silicon Graphics workstation
(Silicon Graphics, Mountain View CA) and examined using
Interactive Data Language–based software (IDL; Research
Systems, Boulder, CO) written by one of the authors
(J.C.M.). The magnetization transfer ratio (MTR) was calculated as a quantitative index of magnetization transfer effect and was used as a surrogate marker of myelination.9,30
Five regions in the white matter (cerebellar white matter, corona radiata, internal capsule, corpus callosum, and centrum
semiovale) and five regions in the gray matter (cerebellar gray
matter, lateral geniculate nucleus, hippocampus, gray matter
of the gyrus lateralis, and caudate nucleus) were examined in
both the left and right side of the brain. Ten regions of interest were averaged in each brain region on each side of the
Preparation of Brain Tissue
Immediately before death, cats were anesthetized and 0.5ml
heparin (1,000U/ml) was administered intravenously. They
were killed with an intravenous overdose of barbiturates, in
accordance with the American Veterinary Medical Association guidelines, followed by intracardiac perfusion with
250ml of 0.9% cold saline, and then 750ml of 10% neutral
buffered formalin. The cerebrum and rostral brainstem were
sectioned (⬇3mm in thickness) in a dorsal plane. Sequential
sections were alternately frozen in dry ice for biochemical
analysis; fixed in 10% formalin for 24 hours, and then paraffin embedded for histology; and fixed in 10% formalin for
24 hours, and then embedded in ornithine carbamoyltransferase and frozen for in situ hybridization. The cerebellum
and caudal brainstem were sectioned transversely into slices
approximately 3mm in thickness and were processed in the
same manner as the forebrain. Paraffin-embedded sections
were stained with either hematoxylin and eosin or Luxol fast
Enzyme Biochemistry for Lysosomal ␣-Mannosidase
Activity in Brain
LAMAN enzymatic activity was measured on formalin-fixed
brain tissue homogenized in Tris buffer. Samples were centrifuged, the supernatant was collected, and enzyme activity
was determined on the substrate 4-methylumbelliferyl-␣-Dmannopyranosidase at 37°C for 1 hour.21,26 Protein determination was done using a modified Lowry method. Activity
was expressed as nanomoles of 4-methylumbelliferone released per milligram total protein per hour in a dorsal plane
section of tissue covering the entire head circumference.
In Situ Hybridization for Feline ␣-Mannosidase
cDNA Messenger RNA
Nonradioactive in situ hybridization was performed on frozen tissue sections to detect fMANB messenger RNA.
Digoxigenin-labeled antisense (700bp) and sense (1,115bp)
complementary RNA probes were generated, and the probe
length was verified on a formaldehyde gel. The in situ hybridization protocol and colorimetric staining using an alkaline phosphatase-mediated nitroblue tetrazolium reduction/
bromochloroindolyl phosphate reaction was performed as
Table. Severity of Neurological Dysfunction in AMD and Treated Cats
8 Weeks
AMD (n)
Treated (n)
Whole-body tremor
Mild (6)
Intention tremor
12 Weeks
18 Weeks
AMD (n)
Treated (n)
AMD (n)
Mild (6)
Moderate (6)
Moderate (6)
Severe (6)
Mild (6)
Mild (6)
Mild (6)
Moderate (6)
Moderate (6)
Severe (6)
Truncal ataxia
Mild (6)
Mild (6)
Moderate (6)
Moderate (6)
Severe (6)
Loss of balance
Absent (6)
Absent (6)
Absent (6)
Absent (6)
Absent (5)
Mild (1)
Absent (6)
Severe (6)
Nystagmus at rest
Absent (4)
Mild (2)
Absent (6)
Euthanasia necessary
Mild (3)
Moderate (3)
Mild (3)
Moderate (3)
Absent (3)
Mild (3)
Absent (3)
Mild (3)
Severe (6)
Treated (n)
All normal cats had no neurological dysfunction at any age.
AMD ⫽ ␣-mannosidosis.
described previously.31,32 Sections from both in situ hybridization and enzyme histochemistry reactions were mounted
on 100% glycerol and photographed using a light microscope.
Statistical Methods
The Kruskal–Wallis test was used to compare whether a significant difference was present among normal, AMD, and
treated cats. If the Kruskal–Wallis test was significant ( p ⬍
0.05), then the Wilcoxon signed rank test was used for pairwise comparison of AMD-normal, AMD-treated, and
normal-treated groups. A p value less than 0.05 was considered significant.
Treated Cats Show Marked Improvement in
Neurological Signs
Cats affected with AMD had a progressive age at onset
of specific signs of nervous system dysfunction. In six
AMD cats, fine whole-body tremors were first seen at a
mean age at onset of 5.0 ⫾ 0.0 weeks. Intention tremors and truncal ataxia developed at 7.3 ⫾ 1.5 and
7.3 ⫾ 2.5 weeks, respectively. Coarse whole-body
tremors (12.0 ⫾ 3.6 weeks) and loss of balance
(13.7 ⫾ 4.2 weeks) had a more variable age at onset of
7 to 16 weeks of age, with all cats showing these signs
of disease by 16 weeks of age. Nystagmus at rest developed at 16 ⫾ 1.0 weeks of age. The cats’ conditions
continued to deteriorate, and they were killed at between 18 and 20 weeks of age.
Six 8-week-old AMD cats were injected with the
AAV1-fMANB vector. These treated cats were injected
intraparenchymally with a total of 125␮l vector into
six tracks in each cerebral hemisphere, plus 25␮l vector
into one track in each cerebellar hemisphere, for a total
of approximately 2.0 ⫻ 1012 genome equivalents per
brain. All cats recovered well and were eating within 12
hours of surgery.
The six cats in the treatment group had disease progression similar to age-matched untreated cats until 12
to 14 weeks of age (Table), when the neurological disease stabilized or improved in every treated cat. Three
cats did not experience development of loss of balance
or a resting nystagmus, and none experienced development of coarse whole-body tremors (see the Table).
From 12 to 18 weeks of age, the intention tremor and
truncal ataxia improved in three cats, and the wholebody tremor improved in all treated cats (see the Table). By 18 weeks of age, all of the treated cats had
only mild clinical signs, and their condition was markedly improved compared with untreated aged-matched
AMD cats. The treated cats could be distinguished
from normal cats only by a mild truncal ataxia, an intermittent fine head tremor, and a short-strided gait.
All of the treated cats required euthanasia by 18 weeks
of age. A video comparing a 16-week-old affected cat
with an age-matched treated cat may be viewed (see
supplemental figure).
Magnetic Resonance Imaging Abnormalities Improve
in Treated Cats
T2-weighted MRI of the brains of 16-week-old cats
with AMD showed the cerebral hemisphere and cerebellar white matter to be isointense to gray matter (Fig
1). In comparison, normal cats showed the white matter to be hypointense to gray matter. Abnormal white
matter signal intensity in AMD cats has been previously shown to be caused by decreased myelination.9
Cats treated with AAV1-fMANB showed the signal intensity of the cerebral white matter to be hypointense
to gray matter (see Fig 1). In the cerebellum, treated
cats showed less of an improvement in signal intensity,
with most regions remaining isointense to gray matter.
The MTR is a normalized value of signal intensity
that allows quantitative comparison of the magnetizaVite et al: Gene Therapy for ␣-Mannosidosis
Fig 1. Normalization of magnetic resonance imaging in adeno-associated virus serotype 1 vector-feline ␣-mannosidase gene (AAV1fMANB)–treated cats. Transverse images of the cerebrum at the level of the thalamus in 16-week-old cats are shown. In normal
cats, white matter is hypointense to gray matter, as can be seen in the corona radiata (arrowhead). In contrast, in cats with AMD,
white matter is isointense to gray matter. In affected cats treated at 8 weeks of age, white matter signal intensity is more similar to
that of normal cats. Magnetization transfer ratio values (mean and standard deviation ) of white matter tracts at 16 weeks of age
in normal, AMD, and AAV1-fMANB–treated cats are shown. AMD ⫽ ␣-mannosidosis.
tion transfer effect, and it can be used as an antemortem quantitative marker of myelination.30 At 8 and 16
weeks of age, AMD cats had an MTR significantly less
than age-matched normal cats, indicating the presence
of significant myelination abnormalities.9 Remarkably,
when treated cats were examined at 16 weeks of age,
their MTR values were statistically the same as normal
cats ( p ⬍ 0.05) in the corona radiata, internal capsule,
and corpus callosum (see Fig 1). Comparison of treated
cats with aged-matched AMD cats showed significant
improvement ( p ⬍ 0.05) in the corona radiata, internal capsule, corpus callosum, and centrum semiovale
(see Fig 1). No significant change was detected in the
cerebellar white matter. In the gray matter, there are
no statistically significant differences in the MTR between normal and AMD cats9; thus, no difference was
expected in the treated cats, which was the case (data
not shown).
Postmortem Analysis
Four of the treated cats were killed at 18 weeks of age
to evaluate brain pathology. One treated cat was evaluated weekly until 30 weeks of age, and one cat was
evaluated until 56 weeks of age. The cat that was killed
at 30 weeks of age had begun to show neurological
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abnormalities similar to those seen in untreated 18week-old cats, except that no whole-body tremors developed; thus, disease progression was significantly delayed. The cat that was killed at 56 weeks of age had
no deterioration in clinical status after 18 weeks of age.
At 56 weeks of age, this cat showed no intention
tremor and had only a mild, intermittent, fine, wholebody tremor; an occasional loss of balance, and a shortstrided gait.
The brains were removed, and the location of the
needle tracks were identified grossly by small indentations on each brain’s surface and histologically by the
presence of hemosiderin (Fig 2A, B). In situ hybridization demonstrated fMANB messenger RNA to be
present up to 2.5mm from the needle track, and evidence of transduction was found in the gray and the
white matter (see Fig 2B). The maximum radius from
the needle track in which in situ–positive cells were
present was measured and averaged for the cerebral
cortex, caudate nucleus, thalamus, hippocampus, and
internal capsule. No significant differences in the radius of transduction were found among these brain regions, and the average of all five brain regions showed
a transduction radius of 1.40 ⫾ 0.20mm. The enzymatic activity of LAMAN was measured on 1-mm-
Fig 2. Increased ␣-mannosidase gene (MANB) messenger RNA and lysosomal ␣-mannosidase (LAMAN) activity in treated cats. (A)
A dorsoventral view of the cat brain shows the injection sites of adeno-associated virus serotype 1 vector-feline ␣-mannosidase gene
(AAV1-fMANB). Transduction of the following specific brain regions was assessed: the frontal cortex and corona radiata at site 1;
the caudate nucleus at site 2; the thalamus at sites 3 and 4; the centrum semiovale and internal capsule at site 5; the hippocampus
at site 6; and the cerebellum at site 7. (B) In situ hybridization with the antisense probe demonstrated MANB messenger RNA in
gray and white matter up to 2.5mm from the injection site. The sense probe, used as a control, showed little to no evidence of hybridization (original magnification, ⫻50; bar ⫽ 1mm). (C) In treated cats (n ⫽ 3), LAMAN activity in a dorsal plane section of
brain covering the entire head circumference was 4% of that found in normal brain (n ⫽ 3) and 7% of that found in the brain
of heterozygote cats (n ⫽ 3).
Vite et al: Gene Therapy for ␣-Mannosidosis
Fig 3. Resolution of neuronal storage seen on hematoxylin and eosin–stained tissue. ␣-Mannosidosis (AMD) cats showed marked
neuronal swelling caused by lysosomal storage throughout the brain. In contrast, treated cats showed complete resolution of cellular
swelling and storage several millimeters away from the injection site and decreased cellular storage in the occipital cortex, which was
15mm caudal to the nearest injection track. Original magnification, ⫻400. Bar ⫽ 0.1mm. (insets) Original magnification,
thick slabs of formalin-fixed frozen brain tissue cut in a
dorsal plane covering the entire brain circumference.
LAMAN activity was measured as 4% of that found in
normal brain (see Fig 2C).
Histological analysis of tissue sections stained with
hematoxylin and eosin showed complete resolution of
storage in neurons, glia, and endothelial cells up to
4.5mm from the injection track (Fig 3). Complete correction of storage was defined as no evidence of cytoplasmic vacuoles in any cell in a ⫻40 field of view.
The radius of the region of brain surrounding the needle track that showed complete resolution of lysosomal
storage was measured and averaged for the cerebral cortex, caudate nucleus, thalamus, hippocampus, and internal capsule. No significant differences in the radius
of complete correction were found among these brain
regions, and the average of these regions showed a radius of complete storage correction of 2.12 ⫾
0.84mm. No significant difference between the radius
of transduction and the radius of complete correction
of storage was found.
Evidence of lysosomal storage increased as the distance from the needle track increased. However, no regions of the treated cat brain showed lysosomal storage
as severe (as determined by evaluation of the number
of cells affected and the foaminess of the cytoplasm) as
that found in untreated AMD cats. Even in regions of
the brain distant from the injection tracks, such as the
occipital cortex, cells were not as swollen as those seen
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in untreated AMD cats (see Fig 3). Resolution of storage also could be seen in cells of the choroid plexus,
ependyma, and meninges (Fig 4).
Abnormalities of myelination also improved in
treated cats. Histological examination of the brains of
18-week-old AMD cats confirmed myelin deficiency,
identified by Luxol fast blue stain intensity, distention,
and splitting of myelin sheaths (Fig 5). In contrast, the
treated cats showed Luxol fast blue staining intensity of
white matter tracts that was more similar to that seen
in normal cats, as well as less splitting of myelin
sheaths (see Fig 5).
Finally, the brain of the 56-week-old treated cat was
examined. In situ hybridization showed fMANB messenger RNA was being produced a year after injection
of the vector. Lysosomal storage was absent to mild,
and myelination appeared normal throughout the brain
(data not shown).
We tested whether an AAV vector could deliver sufficient amounts of MANB complementary DNA into
neurons and glia to engineer cells in situ to serve as
small “enzyme pumps” to release enzyme and crosscorrect neighboring untransduced cells.33 Injection of
AAV1-fMANB into the brains of cats with AMD resulted in transduction of gray and white matter and
increased LAMAN expression in the brain. Complete
resolution of lysosomal storage was seen in the areas
Fig 4. Resolution of cell swelling in nonneuronal cells of the brain. Treated cats showed resolution of cellular swelling and storage
in the choroid plexus and in ependymal, meningeal, and endothelial cells. Original magnification, ⫻400. Bar ⫽ 0.1mm. (insets)
Original magnification, ⫻1,000. AMD ⫽ ␣-mannosidosis.
overlapping the areas of transduction. However, even
in the untransduced cells of the caudal forebrain, cellular storage was much less than in the same brain regions of untreated AMD cats. Two mechanisms may
account for the improvement in cellular storage at large
distances from the transduction sites. First, only small
quantities of LAMAN diffusing from the injection site
are probably sufficient to improve storage in distant
brain regions. Evidence that low levels (⬍5%) of normal enzyme can mediate dramatic decreases in lysosomal storage has been demonstrated in several mouse
models of lysosomal storage diseases.2,3,34 Second, an
alternate method of enzyme distribution may occur,
such as axonal transport or circulation through the cerebrospinal fluid. Axonal transport of the lysosomal enzyme ␤-glucuronidase was demonstrated in the mucopolysaccharidosis VII mouse brain by finding cells
positive for enzyme activity by a histochemical reaction
stain and negative for gene expression by in situ hybridization.35,36 Unfortunately, an equally sensitive histochemical stain for LAMAN enzymatic activity does
not exist; therefore, similar studies could not be performed in the feline AMD model.
Methods to monitor treatment noninvasively will be
critical to the success of gene therapy of the CNS. The
large size of the cat brain allows for the evaluation of
regional correction of neuropathology using MRI. We
previously used MRI to identify and quantify significant myelin loss in the AMD cat brain at 8 and 16
weeks of age compared with normal age-matched cats.9
In normal cats, there is a significant increase in MTR
in the white matter between 8 and 16 weeks of age
corresponding to the time course of myelin maturation. No increase in MTR was seen in AMD cats during this same period, thus AMD prevents the maturation from proceeding normally.9 In contrast, the
AAV1-fMANB–treated cats had MTR values of the
forebrain that were statistically the same as values for
normal cats at 16 weeks of age. The improved myelination seen in treated cats shows that myelination actually increased in treated cats beyond the results at 8
weeks of age. Thus, treatment of AMD cats during the
period of myelin maturation can resolve some myelination abnormalities, even though extensive disease is already present at the time of therapy. Using the MTR
as a surrogate measure of myelination, we noted greater
improvement in the forebrain than in the cerebellum,
but gene transduction was also less extensive in the cerebellum (see Fig 2). Evidence of improved cerebellar
myelination in treated cats was seen by histological
staining, suggesting that MTR is not as sensitive as histological analysis.
Vite et al: Gene Therapy for ␣-Mannosidosis
Fig 5. Effect of adeno-associated virus serotype 1 vector-feline ␣-mannosidase gene (AAV1-fMANB) on white matter pathology.
Luxol fast blue (LB)– and hematoxylin and eosin (H&E)–stained tissue were compared among normal, ␣-mannosidosis (AMD),
and treated 18-week-old cats. AMD cats showed decreased LB staining intensity, increased spacing between myelin sheaths, and
marked oligodendrocyte swelling. In contrast, treated cats showed LB stain intensity more similar to that seen in normal cats, and
complete resolution of cellular swelling and storage several millimeters from the injection site. Original magnification, ⫻25 (LB);
⫻400 (H&E). Bar ⫽ 1mm (LB); 0.1mm (H&E). (insets) Original magnification, ⫻1,000.
The treated cats showed remarkable improvements
in neurological dysfunction, which were first observed
4 to 6 weeks after surgery (12–14 weeks of age), which
is consistent with the increase of AAV vector expression in the mouse brain.35,37 Between 14 and 18 weeks
of age, no worsening of clinical signs were seen,
whereas the conditions of untreated cats deteriorated
significantly during this period. Most importantly, all
of the treated cats showed improvement in clinical
signs rather than just stabilization, indicating that some
signs of disease can be reversed by treatment. In all
cases, the treated cats were doing well at 18 weeks of
age and euthanasia was performed only to examine
brain pathology. Two cats were examined until 30 and
56 weeks of age, which is longer than any untreated
AMD cat had survived in our and other colonies.9,24,26,29,38
The resolution of clinical signs corresponded to the
improvement in histopathological disease. Continuous
action tremors can be caused by neuronal storage and
decreased myelin,38,39 both of which improved in
treated cats. Signs of cerebellar disease, such as loss of
balance, intention tremors, nystagmus, and truncal
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ataxia, also improved in the treated cats, which showed
a corresponding reduction in Purkinje cell storage and
histological improvement in cerebellar white matter
myelination. Resolution of some but not other signs of
neurological dysfunction may be attributed to either
differences in therapeutic effect among various parts of
the nervous system or the age of cats at the time of
surgery. A likely explanation for the absence of coarse
tremors in the treated cats is that treatment was started
before the age at which this tremor developed in untreated cats, thus preventing its onset. In contrast, gene
transfer improved, but did not fully resolve, the truncal
ataxia and head tremor. This may be because of the
incomplete correction of storage in all cells of the brain
or, alternatively, because of an inability to fully reverse
disease that was present before the initiation of therapy. Some of the neuropathology may not be responsive to normal enzyme at the age we initiated treatment, because AMD cats treated with bone marrow
transplantation at 8 to 12 weeks of age continue to
exhibit mild cerebellar signs, despite significant improvements in overall neurological disease.9
This study shows that somatic gene transfer to the
CNS can deliver functioning enzyme directly to deficient brain cells without the high morbidity and mortality rates associated with bone marrow transplantation.40,41 The global improvement in neuropathology
was achieved by a single operation on a brain that is
much closer in size to the human brain than is the
mouse brain and that has an extensively sulcated cortex, suggesting that this may be a clinically feasible approach for human treatment. The cat and infant brain
have, at their widest dorsal plane width, an area of 12.5
and 140 cm2, respectively. Significant correction of
storage was seen in the occipital cortex of treated cats,
approximately 15mm from the nearest injection track.
Assuming that this correction was caused by LAMAN
diffusing from the nearest injection sites, it is hypothesized that three injection tracks in each cerebral hemisphere may be sufficient to mediate significant storage
correction throughout the entire cat forebrain. Assuming similar transduction and enzyme secretion in the
human infant brain, approximately 20 to 30 injections
into each cerebral hemisphere would be needed for correction. However, the use of stronger promoters to increase the total amount of LAMAN secreted from
transduced cells could further reduce the number of
injections necessary to treat the human brain.
This study was supported by the NIH (National Institute of Neurological Disorders and Stroke, K08-NS02032, NS-38690, C.H.V.;
National Institute of Diabetes and Digestive and Kidney Diseases,
DK-07748, M.A.P.; DK-63973, J.H.W.; National Center for Research Resources, RR-02512, M.E.H.).
We thank P. O’Donnell, J. Zweigel, K. Cullen, T. O’Malley, and
the Animal Models Core of the Center for Comparative Medical
Genetics (RR-02512) School of Veterinary Medicine for assistance
with the animal procedures; A. Polesky, B. Chambers, and J. Burns
for assistance in sectioning and processing tissue; D. Aleman for
assistance in video editing; H. Poptani and S. Magnitsky for assistance in image analysis; T. Berg for providing the fMANB complementary DNA; and A. Salvetti and L. Wang for assistance in viral
vector production (DK-47747).
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