original article

© The American Society of Gene & Cell Therapy

Long-term Improvements in Lifespan and Pathology in CNS and PNS After BMT Plus One Intravenous Injection of AAVrh10-GALC in Twitcher Mice Mohammad A Rafi1, Han Zhi Rao1, Paola Luzi1 and David A Wenger1 Department of Neurology, Sidney Kimmel College of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

1

Krabbe disease is an autosomal recessive disorder resulting from defects in the lysosomal enzyme galactocerebrosidase (GALC). GALC deficiency leads to severe neurological features. The only treatment for presymptomatic infantile patients and later-onset patients is hematopoietic stem cell transplantation (HSCT). This treatment is less than ideal with most patients eventually developing problems with gait and expressive language. Several naturally occurring animal models are available, including twitcher (twi) mice, which have been used for many treatment trials. Previous studies demonstrated that multiple injections of AAVrh10-GALC into the central nervous system (CNS) of neonatal twi mice resulted in significant improvements. Recently we showed that one i.v. injection of AAVrh10-GALC on PND10 resulted in normal GALC activity in the CNS and high activity in the peripheral nervous system (PNS). In the present study, a single i.v. injection of AAVrh10-GALC was given 1 day after bone marrow transplantation (BMT) on PND10. The mice show greatly extended lifespan and normal behavior with improved CNS and PNS findings. Since HSCT is the standard of care in human patients, adding this single i.v. injection of viral vector may greatly improve the treatment outcome. Received 12 June 2015; accepted 3 August 2015; advance online publication 8 September 2015. doi:10.1038/mt.2015.145

INTRODUCTION

Krabbe disease or globoid cell leukodystrophy is an autosomal recessive disorder involving the central and peripheral nervous systems (CNS, PNS). Mutations in the galactocerebrosidase (GALC) gene lower activity of the lysosomal enzyme GALC. This enzyme is responsible for the catabolism of certain galactosphingolipids involved in myelination. When GALC activity is too low, the production of stable myelin is impaired (reviewed in refs. 1,2). In addition to patients who present with signs before 6 months of age and die before 2 years of age, older patients, including adults are also diagnosed.1 The current standard of treatment for presymptomatic infantile patients and mildly affected late-onset patients is hematopoietic stem cell transplantation (HSCT).3–5

Infant patients treated by HSCT within 5 weeks of life have significant preservation of cognitive functioning, but suffer from decreased expressive language and increasing ataxia which usually progresses to an inability to walk independently within a few years of treatment.2,5 This may reflect, in part, the lack of correction of the PNS. Several naturally occurring animal models with low GALC activity are also available and have been used for treatment studies. These include mice, dogs, and nonhuman primates.6–9 The twitcher (twi) mouse model has been used for many treatment trials. These include bone marrow transplantation (BMT),10,11 neural and mesenchymal stem cell transplantation,12–15 substrate reduction therapy,16 pharmacological chaperone therapy,17 gene therapy,18–22 enzyme replacement therapy,23,24 antioxidant therapy, and various combinations of these treatments.25–30 Some of these approaches have resulted in a variable extension of life and evidence for improved myelination. None have resulted in a complete correction of the clinical features and pathology associated with this disease. For complete correction of the features of this disease, the treatment must include a source of adequate GALC activity. This disease also has a significant inflammatory component resulting in changes in certain cytokines and chemokines.31–33 These factors also may need to be corrected to result in a completely effective therapy. Significant pathology and clinical features in human patients and animal models are related to defects in myelination in the PNS.34 BMT in young twi mice can only deliver low levels of GALC activity to the CNS and PNS.10,11 However, these mice do live significantly longer than untreated affected mice. Several studies have shown the ability of AAVrh10 to cross the blood–brain barrier as well as infecting many tissues throughout the body.35–37 Studies from this laboratory using AAVrh10GALC injected intracerebroventicularly, intracerebellarly, and intravenously (i.v.) at PND2 in twi mice resulted in significant improvement in weight, lifespan, fertility, neuropathology, and myelination.21 However, signs of hind leg weakness and paralysis eventually presented. When viral administration was limited to a single i.v. injection at PND10 outcomes were better than when a single injection was administered at PND2, however significantly more viral particles were injected at PND10 (2 × 1011 versus

Correspondence: David A Wenger, Department of Neurology, Sidney Kimmel College of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. E-mail: [email protected] Molecular Therapy  vol. 23 no. 11, 1681–1690 nov. 2015

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Synergistic Effect of BMT+AAVrh10-GALC Treatment

Synergistic effect of combined treatment on the lifespan of the twi mice. The median lifespan of untreated twi mice is ~40 days. Tremor usually starts around PND21, hind leg paralysis at

about PND30 and appearance of a hunchback near the terminal stage. As shown previously,22 twi mice receiving a single i.v. injection of AAVrh10 at PND10-12 have little or no tremor and live about 25–30 days longer than untreated twi mice (P < 0.0001). However, two mice of the 20 mice treated this way had greatly extended lives, one living 147 days and one still living at 285 days (Figure 1a). It is interesting that the longest living female mouse treated only by i.v. injection of viral vector is fertile and has had six litters of pups. While the reason for such extended lives compared to other mice receiving the same treatment is not known, it is clear that a single i.v. injection of AAVrh10 at PND10 has a profound effect on the health of twi mice. The average lifespan of 10 twi mice receiving busulfan-based conditioning and BMT transplantation was 77 days, although one lived 149 days. However, remarkable extended lifespan was shown when BMT and viral i.v. injection were combined (Figure 1a). Of the sixteen affected mice subjected to combined therapy, 10 are still living with the oldest one being over 340 days old and six others being older than 150 days. All treated living mice are behaving normally. Two were sacrificed at different ages for analysis while apparently healthy. Four mice were found dead; three of them were active and behaving normally until the end and none had signs similar to untreated twi mice. Of these three mice, one was a treated female that died at PND122 apparently from gastrointestinal complication several weeks after delivering three pups. Two others had a sudden death at PND218 and PND334 without showing any weight loss or deterioration of their condition. The fourth mouse that was found dead at PND178 had increasing tremors before death, but was not in a terminal condition. Less than 5% of the twi mice receiving combined therapy died before 25 days of age and were not included in this study.

a

c

7.6 × 109).22 In addition to longer lifespans, there was significant correction of myelination in both the PNS and CNS.22 It was shown that a single i.v. injection of AAVrh10-GALC at PND1012 resulted in normal levels of GALC in the brain, spinal cord, and cerebellum, supra-normal levels in the sciatic nerves and very high levels in the liver, heart, and muscle. The mice receiving this treatment had an average lifespan of 70 days, no tremor, a normal walking pattern, normal fertility and continued to gain weight until a few weeks before they died.22 However, mice receiving multiple injections into the CNS in addition to i.v. injection on PND2 had a better overall outcome, living on average 104 days.21 It would be ideal if multiple sites for injection of viral vectors would not have to be used in the treatment of human patients. In the current study, we combined BMT at PND9-10 with a single i.v. injection of AAVrh10-GALC 1 day later. The treated affected mice show a remarkable extension of life, little or no tremor, normal walking and strength, maintenance of their weight until the end of life and near normal myelination in the brain, spinal cord, and peripheral nerves. It is proposed that BMT is successful in mitigating some of the inflammatory consequences of this disease while the AAVrh10-GALC provides higher levels of GALC activity to the CNS and PNS. This approach of a single i.v. injection of AAVrh10-GALC following HSCT, the current “standard of care” for human patients, could be used in the treatment of some individuals affected with Krabbe disease.

RESULTS Effects on the lifespan, weight, and behavior

Percent survival

100

iv at PND10 only (n = 20) BMT only (n = 10) BMT + iv at PND 10 (n = 16)

50

Untreated affected (n = 9) 0 0

25

50

75 100 125 150 175 200 225 250 275 300 325 350 Days

b

Wild type

40

BMT BMT + AAV

Weight (g)

30

Untreated

20 10

Wild type

Untreated affected

150-day treated

300-day treated

0 0

10

20

30

40

50

60

Weeks

Figure 1 Effects of bone marrow transplantation (BMT) and BMT + AAVrh10 treatments on lifespan, weight and gait. (a) Survival of the mice treated with BMT and AAVrh10 alone or in combination is compared to untreated affected and wild-type mice. Vertical blue and green upticks represent mice still living, red upticks refer to mice sacrificed for analysis. The asterisk indicates the mouse that died from gastrointestinal complication. (b) Weights of the mice treated with BMT only (n = 10) and BMT + AAVrh10 (n = 16) are compared to untreated affected (n = 9) and wild-type mice (n = 4). (c) Gait analysis. The footprints of mice were recorded as described in the Materials and Methods section. Footprints are from an 80-day-old wild-type mouse, a 42-day-old untreated affected mouse, a 150-day-old, and a 300-day-old combined treated mouse.

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a

b

c

d

Synergistic Effect of BMT+AAVrh10-GALC Treatment

Figure 2 Histochemical detection of galactocerebrosidase (GALC) activity using modified X-gal staining. All tissues are from a 150-dayold twi mouse treated with both BMT and AAVrh10. The blue color represents GALC activity. (a) Cortical region of the brain (original magnification ×100); (b) cross section of the cerebellum (original magnification ×40); (c) lumbar region of the spinal cord (original magnification ×100); and (d) cross section of the sciatic nerve (original magnification ×200).

Cerebellum

Spinal cord

110-day-old wild type

Brain

Physical appearance and growth monitoring. When the initial dose of busulfan was set at 35 mg/kg of body weight a few mice treated with BMT alone or combined with viral injection demonstrated some of the previously reported toxic effects of busulfan myelosuppression including decreased body weight, ruffled fur, and occasional seizures.38,39 Other than decreased body weight compared to wild-type mice, these events have not occurred in mice receiving a busulfan dose of 30 mg/kg. Body weights of the BMT-treated and BMT/AAVrh10-treated mice were monitored as an indicator of general health and compared to the untreated twi and wild-type mice. The untreated twi and wild-type mice had similar weight gain up to ~3 weeks of age (Figure 1b). By week 6 or 7, all untreated mice had died following steady weight loss. Starting around week 2, the BMT-treated mice, with or without viral injection, demonstrated an approximately 15–20% reduction in body weight compared to wild-type mice. While the long-living BMT/AAVrh10 treated mice had lower weights than wild-type mice, these mice maintained their weight for their whole lives (Figure 1b). The lower body weights probably reflect the effects of the busulfan used for myelosuppression. However, the affected mice treated only by BMT gained less weight, compared to affected mice treated by combined therapy (Figure 1b). This demonstrates the better outcome of the mice treated by combined therapy.

b

c

d

e

f

g

h

i

j

k

l

m

n

o

224-day-old BMT + AAV

150-day-old BMT + AAV

98-day-old BMT only

42-day-old Untreated

a

Figure 3 Pathological studies in central nervous system (CNS). Tissues from cerebral hemispheres, cerebellum, and spinal cord from different treated mice are compared to affected untreated and wild-type mice. All images are from paraffin sections stained with luxol-fast blue/periodic acid Schiff. Original magnifications for brain and spinal cords samples are ×600 and for cerebellum is ×400. Myelin is stained blue and infiltrating foamy macrophages are stained pink-red. BMT, bone marrow transplantation.

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Synergistic Effect of BMT+AAVrh10-GALC Treatment

Untreated affected

150-day old BMT + AAV treated

Brain

Wild type

b

c

d

e

f

g

h

i

Spinal cord

Cerebellum

a

Figure 4 Staining of activated microglia/macrophages in CNS of the 150-day-old BMT + AAVrh10-treated mouse compared to the 42-day-old untreated twitcher and 110-day-old wild-type mice. All images are from PFA-fixed frozen sections stained with CD68 antibody. Images (a) through (c) are from cerebral hemisphere white matter (original magnification ×100); images (d) through (f) are from cerebellum (original magnification ×200); images (g) through (i) are from spinal cord white matter (original magnification ×400). As shown in image c and f, brain and cerebellum of the mouse treated with combination of BMT and viral injection, sacrificed at PND150, do not have any CD68-positive cells. However, a section from the lumbar region of the spinal cord does show some staining for CD68 (i). BMT, bone marrow transplantation; CNS, central nervous system; PFA, paraformaldehyde.

Motor function and behavioral monitoring. Head tremor and decreased body weight are the initial clinical features of the twi mice. Weakness in the hind limbs starts after about 4 weeks of age. When lifted by the tail, the symptomatic twi mice do not splay their hind legs as normal mice do, but instead they bring them together clasping their paws. Twitching and the other characteristic features, although reduced in intensity, are still present in the mice treated only by BMT. However, mice receiving combined therapy are free of these signs. The only apparent differences with age-matched wild-type mice are their reduced body size, slight change in coat color, and occasional minor seizures in a small number of mice. All these changes seem to be side effects of the myelosuppressive regimen and are less evident in mice given the lower dose of busulfan. However, it should be noted that human patients receiving HSCT are myelosuppressed using a different drug regimen. Mice treated with the combined therapy demonstrated normal behavior for almost their entire life. This included continuous exploratory movements, climbing the walls of the cage, and walking pattern. Gait was tested in two treated mice at 150 and 300 days of age. As shown in Figure 1c, the gaits of these two mice are essentially normal. Gait of an 80-day-old wild-type mouse and an untreated 42-day-old twi mouse are shown in Figure 1c for comparison. Strength evaluation. Hanging and grip strength test was used to evaluate strength and balance of the treated mice (see Materials and Methods). The grip strength test is an excellent way to appraise the functioning of the peripheral nerves. Untreated twi 1684

a

b

c

d

Figure 5 Astrocyte activation. All images are from PFA-fixed frozen sections stained with glial fibrillary acidic protein antibody that detects astrocytes. Image (a) is from a 110-day-old wild-type mouse, image (b) from 42-day-old untreated twi, image (c) from the 150-day-old BMT + AAVrh10-treated mouse, and image (d) from 224-day-old BMT + AAVrh10-treated mouse (all original magnifications are ×200). As shown in images c and d, the characteristic astrogliosis seen in the untreated twi mouse (image b) appears normal in the combined treated mice. BMT, bone marrow transplantation; PFA, paraformaldehyde.

mice have impaired motor function and are unable to perform any hang test after 3–4 weeks of age. However, mice that received BMT plus a single viral injection, tested between 120 and 310 days www.moleculartherapy.org  vol. 23 no. 11 nov. 2015

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Synergistic Effect of BMT+AAVrh10-GALC Treatment

Wild type

Untreated affected

a

b 150-day BMT + AAV

BMT only

c 224-day BMT + AAV

d

e

334-day BMT + AAV

f

Figure 6 Pathological studies of peripheral nervous system. Cross sections from sciatic nerves of different treated mice are compared to the similar sections from affected untreated and wild-type mice. All images are from paraffin sections stained with luxol-fast blue/periodic acid Schiff. Original magnification for all tissues is ×1,000. The wild-type mouse shows normal myelination (a) while the 42-day-old untreated twi (b) has essentially no myelin and many macrophages. Also the 98-day-old twi mouse treated only with BMT (c) has lost essentially all myelin and is comparable to the untreated affected mouse. In contrast, sciatic nerves from mice of different ages treated with combined BMT/AAVrh10 (d–f) have completely normal looking myelin, and are comparable to the wild-type mouse. BMT, bone marrow transplantation. Wild type

a

e

b

4 µm

f

c

4 µm

334-day-old BMT + AAVrh10

150-day-old BMT + AAVrh10

Untreated affected

g

d

4 µm

h

4 µm

Figure 7 Ultra-structural studies of the peripheral nervous system. (a–d) Toluidine blue stained semi-thin sections of sciatic nerves from a wildtype mouse (a), untreated twi mouse (b), 150-day-old (c) and 334-day-old combined treated mice (d) are shown. Dark and fuzzy-looking axons in the 334-day-old treated mouse seem to be crush artifacts. Pictures from all sections are taken at ×600 magnification. (e–h) Electron micrographs from the same mice described above. The electron micrograph from a wild-type mouse shows a normal appearing density of large and small myelinated axons with intact axoplasm (e). While the image from untreated twi mouse rarely shows myelin structures and is filled with characteristic inclusions (f), the images from 150- and 334-day-old mice present normal looking myelinated axons with no infiltration of endoneurial macrophages (g,h). All original magnifications are ×4,000.

of age, performed the test as well as wild-type mice. All treated mice tested, were able to move about and hang on the inverted screen for at least 90 seconds, the maximum time allowed for wild-type mice.

Biochemical and histochemical analysis GALC activities. Tissues from twi mice had virtually no GALC activity. GALC activity from different tissues of twi mice treated with BMT only were variable but generally similar to the GALC Molecular Therapy  vol. 23 no. 11 nov. 2015

activity of wild-type mice (Table  1). GALC activities in brain, cerebellum, and spinal cord of mice receiving combined ­therapy were comparable to the values obtained from normal mice. However, the sciatic nerves had supra-normal GALC activities (Table 1). In the previous study, we evaluated the GALC activity in the median nerves from the upper limbs of three twi mice treated i.v. with AAVrh10 on PND10-12 and found an average value of 4.6 nmol/hour/mg protein, similar to values measured in sciatic nerves of mice receiving combined therapy (Table 1). 1685

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Synergistic Effect of BMT+AAVrh10-GALC Treatment

Table 1 GALC activity (nmol/hour/mg protein) of BMT and BMT + AAVrh10-treated mice in CNS, PNS, and some peripheral tissues compared to wild-type and untreated affected mice

Brain

WT (32–90-dayold) n = 6

Aff. (35–45-dayold) n = 3

BMT (78, 98, 149-day-old)

1.0–3.6

0

0.3, 1.0, 1.4

0–0.1

BMT + AAVrh10 Died: 112-day-old

3.4

Killed: 150-day-old

2.3

Died: 218-day-old

0.6

Killed: 224-day-old

Died: 334-day-old

1.3

1.2

Cerebellum

1.0–3.4

0.4, 0.8, 1.9

4.0

4.0

1.4

1.3

1.1

Spinal cord

0.8–2.5

0

0.5, 1.4, 1.9

10.5

1.9

4.5

4.1

1.3

Sciatic nerve

0.7–2.0

0.1–0.2

1.6, 1.0, 1.6

7.3

12.0

9.2

9.0

22.1

Liver

1.7–4.1

0.1–0.2

1.5, 1.6

28.2

49.0

15.6

47.0

23.8

Heart

0.4–2.1

0–0.2

4.6, 4.3

139.3

128.3

106.4

111.0

138.0

Skeletal muscle

0.4–0.6

0–0.1

0.9, 1.8

87.2

94.1

82.6

51.9

93.7

BMT, bone marrow transplantation; CNS, central nervous system; GALC, galactocerebrosidase; PNS, peripheral nervous system.

Extremely high GALC activities also were measured in heart, skeletal muscles, and liver (Table 1). Examination of these latter tissues did not reveal any obvious structural abnormalities due to these high GALC values. It is noteworthy to mention that the high GALC expression in tissues of the treated mice was stable throughout their lifespan. GALC distribution. X-gal staining was carried out to localize the GALC activity in different tissues of treated mice. As shown in Figure 2, the CNS had a diffused pattern of staining for GALC activity. However, the Purkinje cells in cerebellum and some neurons in the spinal cord seem to have higher GALC expression (Figure 2b,c). In contrast, the sciatic nerve displays extremely intense X-gal staining (Figure 2d). Intense blue staining of sciatic nerve is in accordance with the supra-normal activity measured when a sample of tissue is homogenized and assayed using radio-labeled natural substrate (Table 1). As shown previously,21 the X-gal staining method does not stain GALC activity in tissues from wild-type mice, ­illustrating the low level of GALC activity present under normal conditions. Very strong blue staining was observed in non-neural t­issues, such as liver, heart, and skeletal muscle of mice that received the combined therapy similar to what was shown previously in twi mice receiving only AAVrh10 i.v. on PND10.22 This finding is in agreement with the high activity measured in these tissues (Table 1).

Pathological assessment Evaluation of the CNS. The myelin integrity and macrophage infiltration status of the treated mice were evaluated by luxol-fast blue/periodic acid Schiff (LFB/PAS) staining of formalin fixed, paraffin embedded sections from different areas in the CNS. As shown in Figure 3, white matter of cerebral hemispheres, cerebellum, and the spinal cord of the 110-day-old wildtype mouse shows strong staining of intact appearing myelin Figure 3a–c. In c­ ontrast to the wild-type mouse, the white matter of the cerebral hemispheres, the cerebellum, and the spinal cord of the 42-day-old untreated twi mouse demonstrates a marked loss of myelin with infiltration of numerous PAS-positive foamy macrophages Figure 3d–f. LFB/PAS staining of the 98-day-old mouse treated with BMT only was similar in appearance to the untreated twi mouse and showed substantial myelin loss and infiltration of numerous PAS-positive foamy macrophages in the cerebral 1686

hemispheres, the cerebellum, and white matter of the spinal cord (Figure 3g–i). The white matter from the CNS of the 150-day-old (Figure 3j–l) and the 224-day-old (Figure 3m–o) mice treated with BMT and AAV, demonstrated much more intact appearing myelin than the untreated twi mouse or the BMT only treated twi mouse. Some variability in staining intensity probably reflects the use of different batches of regents over time. However, only very few PAS-positive foamy macrophages were noted in the cerebral hemispheres and the cerebellum in these mice. However, while strong staining of intact appearing myelin was present in spinal cord white matter of the 150-day-old combined treated mouse, a number of PAS-positive foamy macrophages were also present in this tissue. Figure 4 shows the status of activated macrophage/microglia that can be revealed by anti-CD68 antibody. The CNS of the untreated 42-day-old twi mice shows numerous activated CD68positive cells (Figure 4b,e,h). These activated cells are not present in wild-type mice (Figure 4a,d,g). In contrast to the untreated twi mouse, the brain and cerebellum of the 150-day-old BMT/AAVtreated mouse are free of activated microglial/macrophages and are comparable to the wild-type mice (Figure 4c,f). However, spinal cord of this mouse shows several CD68-positive cells ­ (Figure 4i). Astrogliosis or increased number of the reactive astrocytes is another indicator of disease severity.40,41 In Figure 5, anti-glial fibrillary acidic protein stained brain sections from the 150-day and 224-day-old treated mice are compared to 42-day-old untreated affected and 110-day-old wild-type mice. The brain section of a wild-type mouse shows an orderly arrangement of normal-appearing astrocytes (Figure 5a). In contrast to the wild-type mouse, the untreated twi mouse demonstrates an increased number of hypertrophic reactive appearing astrocytes (Figure 5b). The astrocytic density in the 150-day-old and 224-day-old mice with combined treatment is comparable to the wild-type mouse (Figure 5c,d). Evaluation of the peripheral nervous system. The PNS of twi mice remains untreated by current treatment methodologies. However we have previously shown that i.v. injection of AAVrh10 on PND10 results in high GALC activity in peripheral nerves.22 Therefore peripheral nerves from mice receiving combined therapy were examined for the evidence of pathology. On visual observation, the sciatic nerves of untreated twi mice www.moleculartherapy.org  vol. 23 no. 11 nov. 2015

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are much enlarged compared to wild-type mice due to edema. However, the sciatic nerves are thin and have a normal appearance in mice receiving either AAVrh10 i.v. at PND10 alone or BMT/AAVrh10 therapy. LFB/PAS-stained sciatic nerve from the 110-day-old, wild-type mouse shows strong staining of intact appearing myelin in peripheral nerves (Figure 6a). Near complete loss of myelin is noted in the peripheral nerve of the 42-day-old untreated affected mouse (Figure 6b). Additionally, numerous PAS-positive foamy macrophages are present in the endoneurium of the sciatic nerve of this mouse. The peripheral nerve of the mouse treated only with BMT is similar in appearance to the peripheral nerve from the untreated affected mouse with almost complete loss of myelin (Figure 6c). Numerous endoneurial PASpositive macrophages are also present in the sciatic nerve of this mouse. In contrast, the peripheral nerves from the 150-, the 224-, and 334-day-old mice treated with combined BMT and AAVrh10 show strong staining of the myelin of intact axons (Figure 6d–f). No infiltration of PAS-positive foamy macrophages is identified in the peripheral nerves from any of these three mice. As a further evidence for the correction of the peripheral nerves in twi mice treated by combined therapy, sciatic nerves from these mice were examined by ultrastructural studies. Toluidine blue stained semi-thin sections of sciatic nerve from the wildtype mouse display a normal appearing density of large and small myelinated axons with intact axoplasm (Figure 7a). Comparable sections from the untreated affected mouse show infiltration of numerous endoneurial macrophages and a marked loss of myelinated axons (Figure 7b). Similar sections from the 150-day-old and the 334-day-old combined treated mice show numerous normal appearing myelinated axons. No infiltration of endoneurial macrophages is identified (Figure 7c,d). However, some axons in the 334-day-old treated mouse show crush artifacts (Figure 7d). Electron micrographs from sciatic nerve of the wild-type mouse show normal appearing myelin wrapping on numerous axons (Figure 7e). In contrast to the wild-type mouse, the sciatic nerve of the untreated twi mouse shows infiltration of numerous endoneurial macrophages and a marked loss of myelinated axons (Figure 7f). The ultrastructure of axons and myelin in mice receiving combined therapy are nearly identical to those seen in the wild-type mouse (Figure 7g,h). No infiltration of endoneurial macrophages is identified. Clearly, normal myelination in the sciatic nerve continues throughout the lives of the mice treated by BMT and AAVrh10 i.v. injection.

DISCUSSION

Krabbe disease is a severe lysosomal storage disorder that results from defects in myelination in the CNS and PNS. The only therapy approved for patients with Krabbe disease, namely HSCT performed early in life for infantile patients and milder late-onset human patients, is less than ideal.3–5 While survival is increased significantly, most treated patients develop severe deficits in expressive language and motor functioning requiring aids for mobility several years after treatment. Studies in the animal models of Krabbe disease have been used to explore many treatment options. While some resulted in prolonged lives for the treated affected animals most are not considered satisfactory for human use. Recently we demonstrated that a single injection of AAVrh10-mGALC on Molecular Therapy  vol. 23 no. 11 nov. 2015

Synergistic Effect of BMT+AAVrh10-GALC Treatment

PND10 to presymptomatic twi mice delivered a normal level of GALC activity to the brain and spinal cord and supra-normal activity to the sciatic nerves.22 The treatment also resulted in normal appearing myelination in the brain, spinal cord, and sciatic nerves. The treated mice had no or only minor tremor and normal gait until near the end of their lives. In addition, these mice are fertile, with one female having six litters of pups. While it usually postulated that starting treatment as early as possible is better for most metabolic disorders, including lysosomal diseases, it is interesting that starting treatment around PND10 is more effective than starting at PND2 in the twi mouse.22 This could be for several reasons. A larger volume of vector can be injected at PND10 or possibly very high GALC activity at PND2 may interfere with critical signaling between axons and glial cells during the period when the nervous systems are rapidly developing. It has been shown in cultured oligodendrocytes that very high levels of GALC activity appear to have detrimental effects on differentiating myelin forming cells.42 Although the mice treated at PND10 with AAVrh10 have an extended lifespan and have increased GALC activity, there still seems to be issues not corrected by this approach. Other changes that are found in tissues from patients with Krabbe disease and animal models, including inflammatory factors, may not be corrected by only supplying GALC activity to both the CNS and PNS.31–33 Therefore the present study, combining a single i.v. injection of AAVrh10 one day after BMT, was initiated. The results show dramatic synergy with this relatively simple approach. In previous studies, we and others used irradiation for myelosuppression in mice, however this is no longer used in human patients and therefore we decided to use busulfan in these studies. Busulfan has been used previously for myelosuppression in twi mice by Yeager et al.43 However, we found their method for solubilizing the busulfan to be difficult and therefore switched to using dimethylsulfoxide for this purpose. Unknown to us another study used dimethylsulfoxide to solubilize busulfan for use in BMT in mice without complications.44 We found a dose of 30 ml/kg body weight injected i.p. on PND8 or 9 into wild-type mice and twi mice to be efficient in eliminating host bone marrow cells. Twentyfour hours later, the busulfan-treated mice received a BMT. Some mice receiving only BMT were followed and they had an ­average lifespan of 77 days, although a few lived longer (Figure 1a). Other twi mice received a single i.v. injection of AAVrh10 on the day following BMT. Those mice with combined therapy lived much longer than mice that received BMT alone or vector only injected i.v. on PND10 (Figure 1a).22 The mice treated with combined BMT/AAVrh10 had either no tremor or only very slight tremor and they maintained their weight throughout their lives (Figure 1b). Similar to what was reported previously22 in mice injected i.v. on PND10 with AAVrh10 there was normal GALC activity in brain and spinal cord and supra-normal levels in sciatic nerves (Table 1). While a small amount of GALC activity is supplied by the BMT, more, especially in the sciatic nerve, was supplied by the vector. As shown in Table 1 and by X-gal staining, i.v. injection of this vector results in a very wide tissue distribution confirming previous studies by us and others.21,22,35–37 As GALC is a typical lysosomal enzyme, it is able to be secreted and taken up by neighboring cells eliminating the need to transduce every cell 1687

Synergistic Effect of BMT+AAVrh10-GALC Treatment

to instill pathological correction.45 Therefore, looking for expression of the transgene in critical tissues instead of vector was more relevant to this study. This study confirms that iv-injected AAVrh10 is able to provide high GALC activity to many tissues including peripheral nerves. The ability of this vector to deliver a corrective gene product to the PNS is especially important considering that most human patients treated by HSCT have significant issues with peripheral neuropathy manifesting years after treatment. The fact that these mice have normal gait until near the end of their lives, indicates the positive effect that this combined treatment can have. When i.v. injection of AAVrh10 is combined with BMT, the extension of life for affected twi mice is very dramatic. Since the majority of the treated mice are currently still alive, it is not possible to calculate an average lifespan. However, most of them have survived beyond 200 days and are still healthy. This result is a clear indicator of the synergistic effect of the combined therapy. While it is obvious that this treatment results in improved myelination in all nervous tissues, some activated macrophages and microglial cells remain in certain tissues, especially in the spinal cord (Figures 3 and 4). This was shown by both LFB/PAS staining and immunostaining for CD68. This may reflect early pathological damage not cleared by the GALC activity provided or insufficient anti-inflammatory factors from the hematopoietic cells of the donor. However, it is obvious that combined therapy is better than BMT alone in restoring myelination and decreasing the number of PAS-positive cells. As stated earlier, correction of the PNS in both human patients and animal models has remained difficult until now. With the combination of BMT and viral gene therapy, the mice lived much longer and they maintained high GALC expression in all tissues, especially in the sciatic nerve, with normal myelination even in the mouse that lived 334 days (Figures 6 and 7). This can be explained by supra-normal GALC activity in peripheral nerves of the combined treated mice and illustrates the high tropism of AAVrh10 toward the PNS.22 These results indicate that adding a single i.v. injection of AAVrh10 containing the human GALC cDNA following HSCT may lead to a significant increase in lifespan with an improved quality of life in human patients with Krabbe disease. Although many lysosomal storage disorders have a white matter component to their pathology, globoid cell leukodystrophy, and metachromatic leukodystrophy are considered primary demyelinating lysosomal disorders. Gene therapy approaches for the treatment of metachromatic leukodystrophy are now in clinical trials.46,47 Not adequately addressed by these procedures is delivery of arylsulfatase A activity to the PNS which is significantly involved in the pathology of patients with metachromatic leukodystrophy. Therefore, the i.v. injection of this vector containing the appropriate gene could be used to treat other genetic disorders needing delivery of a gene product to the PNS. In conclusion, the combination of BMT and a single i.v. injection of AAVrh10-GALC around PND10 can result in a dramatic increase in lifespan of twi mice. These mice have near normal myelination in the CNS and PNS, show less pathological features of this disease and exhibit normal behavior. Further studies are planned related to the timing of viral administration after BMT and the dosing needed to achieve maximum survival. Studies 1688

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on the larger canine model are also in progress. It is hoped that this approach can lead to a clinical trial in human patients with Krabbe disease in the near future.

MATERIALS AND METHODS

Generation of AAV2/rh10-mGALC vector. Generation of viral construct used in this study has been previously reported.21 Briefly, pCB7plasmid, an enhanced version of AAV2 vector was received from the Institute for Human Gene Therapy at the University of Pennsylvania. Mouse GALC cDNA was cloned into EcoRI site of this construct, downstream from the human CMV-enhancer/chicken β-actin hybrid promoter. The accuracy of the transgene was confirmed by sequencing, the integrity of the 5′and 3′ ITRs was confirmed by restriction enzyme analysis and the functionality of the construct was verified by in vitro cell transfection and measurement of GALC enzyme activity. Plasmid Maxi kit from Qiagen (Valencia, CA) was used for large-scale plasmid preparation which was cross-packaged into AAVrh10 capsid by utilizing a chimeric AAV2-Rep/AAV1-Cap and helper plasmids during a triple-transfection procedure.48,49 Viral packaging and purification were accomplished by the Institute for Human Gene Therapy at the University of Pennsylvania. The viral titer determined by polymerase chain reaction of the simian virus 40 poly(A) sequence48,49 was 8.1 × 1012 genomic equivalents/ml. Animal procedures. Twi mice were originally obtained from the Jackson

Laboratory. These mice, with W339X mutation in the GALC gene, are in the C57BL background. Affected mice obtained from heterozygous matings were genotyped on PND1 or 2. Toe clips were used for both mouse identification and DNA extraction for genotyping. Genotyping of newborn mice was determined by polymerase chain reaction and restriction enzyme analysis.22,50 As male and female twi mice had identical features, mice of both sexes were used in these studies, and no differences in outcomes following treatment were noted. Treated mice were observed daily during the weekdays, and if deemed moribund, (inactive with severe twitching or tremor and weight loss), were killed by CO2 euthanasia and the age of death was recorded. Some mice were sacrificed to obtain tissues for study at different ages although they appeared neurologically completely normal. Other treated mice died of health complications apparently unrelated to the disease or the treatment. Mice breeding, treatment procedures and euthanasia procedures were approved and carried out according to the guidelines of the Institutional Animal Care and Use Committee at Thomas Jefferson University. Bone marrow transplantation. Myelosuppression of mice was achieved by the use of busulfan. A 3 mg/ml solution of busulfan (Sigma, St. Louis, MO) was prepared by dissolving the busulfan in dimethylsulfoxide (30% of the final volume), and adding the remaining volume of sterile phosphate-buffered saline. Eight or 9-day-old affected mice (this small variability in injection times was due to the timing of births) were weighed, and 30 mg/kg of body weight of the busulfan solution was injected intraperitoneally (i.p.). Bone marrow cells from noncarrier, syngeneic mice were obtained by flushing tibiae and femora using ice-cold Hepes buffered Hanks’ balanced salt solution (Mediatech, Manassas, VA) on ice. The cells were then counted, centrifuged, and resuspended in Dulbecco’s modification of Eagle’s medium. Twenty-four hours after busulfan injection mice received an i.p. injection of 3–4 × 107 bone marrow cells in a total volume of 0.2 ml. For at least 2 weeks after BMT, the mice were provided with prophylactic Neomycin (final concentration 500 μg/ml) (Sigma) in drinking water. Transplanted animals were observed daily or at least three times a week for any signs related to the procedure and later for tremor, weakness, and progression of disease. Body weight was recorded weekly throughout their lives. Treated mice were allowed to survive as long as they could be humanely maintained or euthanized using CO2 at specific time points www.moleculartherapy.org  vol. 23 no. 11 nov. 2015

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for biochemical and pathological studies. While some mice received only BMT others also received viral vector. Viral delivery. In the previous study, affected twi mice were given AAVrh10-GALC on PND10-12 without BMT.22 In the present study, viral vector was delivered by a single i.v. injection in the tail vein 24 hours after BMT. Trypan blue was added to the viral stock at a final concentration of 0.05% (wt/vol) to facilitate visual assessment of the injections. The animals were cryo-anesthetized on ice before the injections. A total of 2 × 1011 viral particles in ~25 μl were injected i.v. into the tail vein using a 28G1/2 insulin syringe. This is equivalent to about 4 × 1013 viral particles/kg body weight. Injections were carried out on a light box to facilitate visualization of the tail vein. The success of the correct injection was evaluated by the presence of a blue color in the vein. After the injection, the pups were warmed and returned to their mother. Mice that died within a few days of the injection (less than 5%) were not included in this study. Behavioral studies. Mice were weighed weekly and examined daily during

weekdays for any signs of tremor, weakness, or gait disturbance. In addition, individual mice of different ages were subjected to evaluation of their walking patterns and ability to hang upside down from a wire screen.

Walking pattern. A blind tunnel 34 cm long, 4 cm wide, and 3 cm high was made from a polyvinyl chloride pipe. The front paws of the mice were dipped in nontoxic red food color, and back paws in blue food color. The mouse was placed at the end of the tunnel on the paper so that it could walk down the tunnel towards the light source. The piece of paper with the colored paw prints was retained for comparative analysis. An 80-day-old normal mouse and 42-day-old untreated affected mouse were tested for comparison. Hanging test for strength, balance, and motor skills. Treated mice of different ages were placed on the inside of the top of a box with a wire screen, and the top was closed leaving the mice hanging upside down. The box was 25 cm deep and the bottom was covered with one inch of foam to protect the mice if they fell. Timing was started upon closing the top. Normal mice of different ages were able to hang and move around for at least 90 seconds. Affected mice of different ages treated with BMT plus AAVrh10 were subjected to the same test. The ability to hang on and move around for 90 seconds was considered normal. Untreated twi mice beyond 25 days of age were unable to hang for any length of time. Tissue preparation for microscopic analysis. At the time of sacrifice, the

mice were subjected to transcardiac perfusion. The treated and untreated mice were deeply anesthetized using pentobarbital sodium (100 µg/g body weight) and perfused initially with 40 ml of ice-cold PBS, followed by perfusion with 40 ml ice-cold 4% paraformaldehyde in PBS (PFA/ PBS). Different tissues from CNS and PNS along with tissues from other peripheral organs were removed, postfixed in fresh 4% PFA/PBS for additional 4 hours at room temperature, and cryoprotected by soaking in 25% glucose (w/v) for 18–24 hours at 4 °C until the tissues sank to the bottom of the sucrose solution. The cryo-protected tissues then were embedded in Tissue-Tek optimum cutting temperature compound (Sakura Finetek, Torrance, CA) and frozen in a liquid nitrogen-chilled isopentane (Sigma) bath. The tissue blocks were stored at −80 °C until sectioning. Six-micrometer-thick coronal sections were prepared using an HM-505N Microm cryostat (Richard-Allan, Kalamazoo, MI). Recovered sections on the glass slides were processed directly for staining or stored at −80 °C. Alternatively, tissues from the PFA/PBS perfused mice were post fixed in 10% formalin (3.7% formaldehyde) and processed for paraffin embedding and sectioning. Using the standard procedures, paraffin sections were prepared and stained with LFB/PAS and examined by light microscopy.

Tissue processing for ultrastructural analysis. Sciatic nerves from twi and

control mice were removed from anesthetized mice and submerged into 2% glutaraldehyde in 0.1M phosphate buffer, pH 7.4, followed by postfixation in 1% osmium tetroxide in 0.1M phosphate buffer. The tissue Molecular Therapy  vol. 23 no. 11 nov. 2015

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fragments were dehydrated in series of graded ethanol and infiltrated with mixtures of embedding medium (Spur) and ethanol, utilizing the microwave processing technique. The tissue fragments were embedded in Spur embedding medium and polymerized over night at 80 °C. Semithin (0.5 micron thick) sections were cut on a UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany), using glass knives. The sections were transferred to glass microscope slides, stained with Toluidine Blue O stain, and reviewed using a light microscope. For ultrastructural studies, the 100 nm thick sections were cut using the UC7 ultramicrotome with a diamond knife. Sections were collected on to copper grids and stained with uranyl acetate and lead citrate. These sections were evaluated on a JEOL 100CX II electron microscope. Digital images were created using an Advantage HR Digital CCD Camera System attached to the electron microscope. Tissue preparation for GALC assay. Tissues from treated and control mice were removed immediately after CO2 euthanasia. The tissues were quickly frozen and stored at −80 °C or homogenized in distilled water using a Polytron apparatus (Brinkmann Instruments, Westbury, NY). Protein concentration was determined according to the method of Lowry et al.51 GALC activity was measured using [3H]galactosylceramide substrate, according to our published method.52 GALC activity was expressed as nmol hydrolyzed/h/mg protein. Immunohistochemistry. Frozen sections from different tissues were thawed and fixed for extra 15 minutes with freshly made 4% PFA/PBS at room temperature. These tissues were permeabilized with ethanol/acetic acid (95/5) for 10 minutes at −20 °C, and treated with blocking reagent from Vector Laboratories (Burlingame, CA) for 1 hour. Tissues were then incubated with the first antibody in a humidified chamber at 37 °C overnight. Antibodies included rabbit anti-glial fibrillary acidic protein (Abcam, Cambridge, MA) for astrocytes and rat anti-CD68 (BioRad, Hercules, CA) for macrophages/ microglia. The targeted antigens were visualized by incubating the sections with the secondary anti-rabbit or anti-rat antibodies (Alexa 488 from Molecular Probes, Eugene, OR) for 2 hours at 25 °C. Immunostained slides were mounted with Vectashield mounting medium containing DAPI as a nuclear marker (Vector Lab, Burlingame, CA). Although coronal sections from the entire brain were monitored, most sections studied were from the area between the bregma and interaural lines. Histochemical staining for GALC enzyme activity. Freshly prepared or

f­rozen sections were equilibrated with citrate/phosphate buffer (pH 4.5) for 15 m ­ inutes, and then incubated in 5 mg/ml taurodeoxycholic acid and 5 mg/ ml oleic acid in C/P buffer. X-gal staining was done according the method described by Dolcetta et al.53 Sections were dehydrated in graded solutions of ethanol-water, cleared in Histoclear (Fisher Scientific, Pittsburgh, PA), covered with Permount (Fisher Scientific) and examined under light microscopy. Fluorescence and bright-field microscopy. Olympus BX51 microscope

with FITC, TRITC, and UV filters (Chroma Technology, Brattleboro, VT) was used to visualize the fluorescent and colorimetric-stained slides. Digital images were captured with a SPOT-RT camera (Diagnostic Instruments, Sterling Heights, MI). The compiled images were processed with Adobe Photoshop CS6 software (Adobe Systems, San Jose, CA).

Statistical analysis. GraphPad Prism software was used to determine the

statistical significance. All data are depicted by the magnitudes of means and their related standard error. Comparisons between two groups were performed using two-tailed t-test.

ACKNOWLEDGMENTS This research was supported in part by a grant from The Legacy of Angels Foundation. The authors thank Mark Curtis (Department of Pathology, TJU) for his technical assistance in light microscopy and valuable inputs in interpreting pathological and ultrastructural data. The authors declare no conflict of interest.

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