Long-term amelioration of feline Mucopolysaccharidosis VI after AAV-mediated liver gene transfer

Abstract

Mucopolysaccharidosis VI (MPS VI) is caused by deficient arylsulfatase B (ARSB) activity resulting in lysosomal storage of glycosaminoglycans (GAGs). MPS VI is characterized by dysostosis multiplex, organomegaly, corneal clouding, and heart valve thickening. Gene transfer to a factory organ like liver may provide a lifetime source of secreted ARSB. We show that intravascular administration of adeno-associated viral vectors (AAV) 2/8-TBG-felineARSB in MPS VI cats resulted in ARSB expression up to 1 year, the last time point of the study. In newborn cats, normal circulating ARSB activity was achieved following delivery of high vector doses (6 × 10(13) genome copies (gc)/kg) whereas delivery of AAV2/8 vector doses as low as 2 × 10(12) gc/kg resulted in higher than normal serum ARSB levels in juvenile MPS VI cats. In MPS VI cats showing high serum ARSB levels, independent of the age at treatment, we observed: (i) clearance of GAG storage, (ii) improvement of long bone length, (iii) reduction of heart valve thickness, and (iv) improvement in spontaneous mobility. Thus, AAV2/ 8-mediated liver gene transfer represents a promising therapeutic strategy for MPS VI patients.

Figure 1

Serum ARSB activity levels in MPS VI cats treated with AAV2/8-TBG-fARSB. MPS VI cats were injected either at (a) postnatal day 5 (p5) or at (b–e) p50 with various doses of AAV2/8-TBG-fARSB (as reported inside each panel). Serum ARSB activity was monitored for 12 months after vector administration. Each curve represents ARSB values over time from a single animal. In cats injected at (a) p5 ARSB activity levels similar to those measured in normal cats (NR) were obtained in animals receiving 6 × 10¹³ genome copies (gc/kg) of AAV2/8-TBG-fARSB vectors. In animals injected at (b–e) p50, ARSB activity levels similar or above NR were achieved more consistently in the 6 × 10¹³–2 × 10¹² gc/kg vector dose range. AF, untreated MPS VI cats; °, animals receiving AAV vector constructs containing the WPRE element. Arrows point at the time point of AAV vectors injection. The number of animals analyzed in each group is reported inside each panel. AAV, adeno-associated viral; ARSB, arylsulfatase B; MPS VI, mucopolysaccharidosis VI; TBG, thyroxin binding globulin.

Figure 2

Reduced urinary GAG excretion and mitral valve thickness in MPS VI cats treated with AAV2/8-TBG-fARSB. (a) Urinary GAG levels were measured in 12-month-old normal (NR, white bar), affected (AF₁, black bar) and MPS VI cats receiving different AAV2/8-TBG-fARSB vector doses (reported under each bar) either at p5 or at p50. Urinary GAGs were reduced in animals receiving AAV compared to AF. Stronger reduction was obtained in animals receiving AAV vector doses between 6 × 10¹³ and 6 × 10¹² gc/kg (p50) or 6 × 10¹³ gc/kg (p5). (b) Heart ultrasound analysis of 10-month-old NR, AF, and MPS VI cats treated with AAV vectors. Mitral valve thickness (MVT, subjective score) was significantly increased in AF compared to NR cats. MVT reduction was obtained in cats treated with AAV2/8-TBG-fARSB, independent of the age at treatment and of the AAV vector dose injected. Results are reported as mean ± SE when n > 1; *P value ≤0.05. The number of animals analyzed in each group (n=), the vector doses used and the age at treatment are reported under each bar. AAV, adeno-associated viral; fARSB, feline arylsulfatase B; GAG, glycosaminoglycan; gc, genome copies; MPS VI, mucopolysaccharidosis VI; p5, cats injected at postnatal day 5; p50, cats injected at postnatal day 50; TBG, thyroxin binding globulin.

Figure 3

Improvement of bone size and pathology in MPS VI cats receiving AAV2/8-TBG-fARSB. (a) Femur length in MPS VI cats receiving various doses of AAV2/8-TBG-fARSB vectors. Radiographs were obtained on 10–12-month-old normal (NR), affected (AF), and MPS VI cats treated with AAV vectors and femur length was measured. The femur length, reported as percentage of age- and sex-matched NR cats, was significantly reduced in AF cats and was increased in most animals, independent of the age the cat at treatment. Results are reported as mean ± SE when n > 1; *P value ≤0.05. The number of animals analyzed in each group (n =), the vector doses used and the age at treatment are reported under each bar. White bar: normal cats; black bars: affected cats. (b,c) Representative pictures of (b) femur growth plate and (c) cortical bone histology from NR, AF, and MPS VI cats treated with AAV vectors. GAG storage, evident as vacuoles in cortical bone osteocytes (c, white arrows) and in the growth plate resting zone chondrocytes (b, black arrows) of AF cats was reduced in MPS VI animals receiving AAV vector doses between 6 × 10¹³ and 2 × 10¹² gc/kg (p50) or 6 × 10¹³ (p5). Cats receiving lower vector doses showed only a minor reduction in osteocyte storage. Black lines in each panel delineate the growth plate. Magnification: ×10 for growth plate, ×40 for cortical bone. AAV, adeno-associated viral; fARSB, feline arylsulfatase B; GAG, glycosaminoglycan; gc, genome copies; MPS VI, mucopolysaccharidosis VI; p5, cats injected at postnatal day 5; p50, cats injected at postnatal day 50; TBG, thyroxin binding globulin; WT, wild type.

Figure 4

Improved facial dysmorphism in MPS VI cats treated with AAV vectors. Representative pictures of muzzles from control and MPS VI cats receiving AAV2/8-TBG-fARSB at p5 or at p50. Affected cats (AF) showed flattened muzzle and shortened ear (arrows) when compared to normal (NR) animals. Animals receiving high AAV vector doses [6 × 10¹³–2 × 10¹² gc/kg at p50 (AAV-p50) or 6 × 10¹³ gc/kg at p5 (AAV-p5)] showed amelioration of both ear and muzzle appearence. AAV, adeno-associated viral; gc, genome copies; MPS VI, mucopolysaccharidosis VI.

Figure 5

Improved motor activity in MPS VI cats treated with AAV2/8-TBG-fARSB. (a) Evaluation of spontaneous motor activity in 9–12-month-old control (NR and AF) and AAV vector-treated MPS VI cats. A motor activity score was assigned to each animal based on the ability to spontaneously walk, run, step up, or jump (see Materials and Methods section for details). Animal mobility was significantly reduced in affected cats (AF, black bar) compared to normal (NR, white bar). Higher scores, albeit not significantly different from AF, were observed in cats receiving AAV vector doses between 6 × 10¹³ and 2 × 10¹² gc/kg (p50) or 6 × 10¹³ gc/kg (p5). (b) Evaluation of walking score in 9–12-month-old control and AAV vector-treated MPS VI cats. A walking score was assigned to each animal based on the ability to spontaneously walk with a normal posture; this is significantly reduced in AF as compared to NR and significantly improved in cats receiving high vector doses. Results are reported as mean ± SE when n > 1; *P value ≤0.05. The number of animals analyzed in each group (n=), the vector doses used and the age at treatment are reported under each bar. AAV, adeno-associated viral; AF, affected cats; fARSB, feline arylsulfatase B; GAG, glycosaminoglycan; gc, genome copies; MPS VI, mucopolysaccharidosis VI; NR, normal untreated cats; p5, cats injected at postnatal day 5; p50, cats injected at postnatal day 50; TBG, thyroxin binding globulin.