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Background: Pompe disease (PD) is a rare neuromuscular disorder caused by deficiency of acid alpha-glucosidase (GAA), a lysosomal glycogen-catabolizing enzyme. Despite availability of a recombinant human GAA enzyme replacement therapy (rhGAA ERT), clinical unmet needs remain, including suboptimal responses in skeletal muscles caused in part by several key challenges: instability of ERT in circulation and inefficient uptake via the cation-independent mannose 6-phosphate receptor (CI-MPR) at low interstitial concentrations. Once inside cells, GAA requires processing to attain maximal activity for glycogen degradation; however, the relative contributions of proteolytic and N-glycan processing are poorly understood. AT-GAA—an investigational, 2-component therapy comprising cipaglucosidase alfa (a next-generation rhGAA enriched with bis-phosphorylated N-glycans for improved uptake) administered with miglustat (a small molecule stabilizer of cipaglucosidase alfa)—has been demonstrated to significantly improve the PD pathogenic cascade (eg, glycogen reduction, reversal of autophagic dysfunction, and muscle pathology) compared to alglucosidase alfa in Gaa knockout (KO) mice. We demonstrate that N-glycan processing is required for enzyme activation and further describe the relative impact of the 2 components of AT-GAA on observed efficacy in Gaa KO mice.
Objectives: To evaluate rhGAA and modified rhGAAs resistant to N-glycan trimming for processing and enzyme activation. To further characterize the relative effect of each of the individual components of AT-GAA (cipaglucosidase alfa and miglustat) on observed efficacy in Gaa KO mice.
Results: Cipaglucosidase alfa was fully processed, activated, and indistinguishable from mature, endogenous human GAA; GAA variants resistant to N-glycan trimming demonstrated lower activity. In Gaa KO mice, miglustat stabilized cipaglucosidase alfa and preserved its activity in the unfavorable physiological pH of blood following infusion.
Conclusion: Results highlight the importance of improving both rhGAA ERT uptake and preserving intracellular processing to maximize glycogen degradation. In Gaa KO mice, the impact of miglustat on cipaglucosidase alfa stability and activity is demonstrated, which has relevance toward developing an effective treatment for PD.
Enhancing Delivery of Acid Alpha-Glucosidase (GAA) to Skeletal Muscle in Pompe Disease (PD): Key Challenges and Attributes of AT-GAA
Nithya Selvan1, Suresh Venkateswaran1, Jessie Feng1 , Finn Hung1 , Matthew Madrid1 , Nickita Mehta1 , Matthew Graziano1, Nastry Brignol1, Yuliya McAnany1, Ritchie Khanna1 , Su Xu1, Steven Tuske1, Jon Brudvig2, Hung Do1*
1Amicus Therapeutics, Inc. Philadelphia, PA, USA.2Sanford Research, Sioux Falls, SD, USA.
*Corresponding author – [email protected]
Abstract
Dissecting the effect of protein and glycan processing on GAA activation
Schematic of the chemical modification of sialic acids on rhGAA
Glycan and protein processing are essential for optimal GAA activity
E. Pompe patient fibroblasts were incubated with 500 nM unmodified rhGAA, oxidized rhGAA or rhGAA-AOAA for 16 hours at 37°C. Cells were then incubated further in growth media lacking rhGAAs and harvested at indicated time points, lysed and analyzed by Western blotting. RhGAA and modified rhGAAs were differentially processed following internalization as indicated by banding pattern of intermediate forms of the protein.
F. To differentiate between proteolytic and glycan processing , PNGaseF digests were performed to remove all glycans on lysates harvested at 24-hours. Banding patterns indicated that modification of rhGAA blocks glycan processing of modified structures in cellulo though proteolytic processing still occurs.
Oxidized rhGAA and rhGAA-AOAA can still undergo proteolytic processing but not glycan trimming in cellulo.
Conclusions
Dissecting the effect of miglustat on AT-GAA efficacy
Acknowledgements, funding and conflicts of interest
We wish to thank Yi Lun, Adriane Schilling, Anju Nair, Rebecca Soska, M. Osman Sheikh, Nicholas Siano and Renee Krampetz fortheir contributions to the experiments described here. This work was performed at and funded by Amicus Therapeutics Inc. Allauthors, except JB, are current or former employees of Amicus Therapeutics Inc. and hold equity in the company.
Modified rhGAAs deficient in glycan trimming or proteolytic cleavage serve as tools to dissect the effect of the two forms of processing on the efficiency of glycogen degradation by GAA.
Blocking N-glycan trimming leads to only partial activation of GAA. Both proteolytic cleavage and glycan processing are essential for optimal glycogen degradation by GAA.
AT-GAA is a two-component investigational therapy comprised of cipaglucosidase alfa, which is a form of GAA enriched in natural bis-phosphorylated N-glycans for improved uptake into cells, and miglustat, a small molecule chaperone that stabilizes GAA. Like endogenous GAA, cipaglucosidase alfa undergoes full processing within cells.
Cipaglucosidase alfa is the primary contributor of AT-GAA efficacy. While co-administration of miglustat with cipaglucosidase alfa improves treatment, outcomes compared to administration of cipaglucosidase alone, miglustat alone is not sufficient to ameliorate cellular pathologies associated with PD in Gaa KO mice.
A. 4MU-α-Glc is a small synthetic substrate commonly used to assay for GAA activity. GAA 4MU-α-Glc hydrolytic activity was not affected by chemicalmodifications of N-glycans, removal of N-glycans or GAA proteolytic processing.
B. Glycogen hydrolytic activity of precursor rhGAA or fully processed rhGAA compared to the activity of partially processed rhGAA-AOAA (onlyproteolytically processed) or rhGAA-kif (only deglycosylated – site 5 close to active site is fully deglycosylated). Fully processed rhGAA, but notpartially processed forms, showed improved catalytic efficiency compared to precursor unmodified rhGAA. Error bars represent SEM.
C. Unlike partially processed modified rhGAAs, fully processed rhGAA had statistically significant ((p<0.05) one-way ANOVA with Fisher’s LSD post-hoctest) ~4-fold reduced Km for glycogen.
Blocking GAA glycan processing impairs glycogen hydrolytic activity
57 952
N-14
0
N-23
3
N-39
0
N-47
0
N-65
2
N-88
2
N-92
5
57 952
N-14
0
N-23
3
N-39
0
N-47
0
N-65
2
N-88
2
N-92
5
rhGAA
rhGAA - kif
57 952
N-14
0
N-23
3
N-39
0
N-47
0
N-65
2
N-88
2
N-92
5
rhGAA – kif + Endo H
Not Found
Not Found
Glycopeptide did not ionize sufficiently
Glycopeptide did not ionize sufficiently
40-50% site occupancy
40-50% site occupancy
40-50% site occupancy
rhGAA-kif can be deglycosylated without any proteolytic cleavage
rhGAA - kif
rhGAA – kif + Endo H Key: Hexose HexNAc
GlcNAcMannosePeptide
631ILQFNLLGVPLVGADVCGFLGNTSEE656
y2-H
2O y2y3 y4 b6
y6+H
exN
Ac
y7+H
exN
Acb9
y9+H
exN
Ac
b12
y12+
Hex
NAc
y14+
Hex
NAc
b62+ b11
y10+
Hex
NAc
b12
b6
b7
b9
b11
b12
Hex
NAc
1Hex
2
Hex
NAc
1Hex
3
Hex
NAc
1Hex
4
Hex
NAc
1Hex
5
Hex
2 Hex
NAc
1Hex
6
Hex
NAc
1Hex
7
Hex
NAc
1Hex
8
b12y1
4+H
exN
Ac
y17+
Hex
NAc
Modified rhGAAs undergo proteolytic cleavage but not glycan trimming
Glycopeptide analysis of unmodified rhGAA (C.) and modified rhGAA (D.) with representative MS1 and MS2 spectra of the highly abundant trypticglycopeptide, 882NNTIVNELVR891. Modified sialic acids on rhGAA - AOAA were insensitive to neuraminidase activity.
rhGAA - unmodified
Intermediate 95 kD
WB: GAA
WB: Actin
MW (kD) 15010075
5037
25
5037
Processed
rhGAA - AOAArhGAA - oxidized
Intermediate 76 kD
Processed
Precursor 110 kD
Processed
Precursor Processed – 24 h
unmod
ified
oxidi
zed
AOAAun
modifie
dox
idize
dAOAA
untre
ated
oxidi
zed
AOAA
unmod
ified
PNGaseF - - - - - - - + + + +MW (kD)
15010075
5037
5037
WB: GAA
WB: Actin
Intermediate 95 kD
Intermediate 76 kDDeglycosylated intermediate
Deglycosylated intermediate
Precursor 110 kD
Proteolytically cleaved peptide associated with mature 70kDDeglycosylated cleaved peptide
25201510
untre
ated
A Modified rhGAA that undergoes glycan trimming but not proteolytic cleavage
150100
MW (kD)150
100
Bacterial neuraminidase - + - + - + - +
EB : WGA
WB: GAA
N-glycan trimming as well as proteolytic cleavage are required for full GAA activation
Cipaglucosidase alfa undergoes full processing in cellulo
10075
15
50
37
50
37
250150
25
MW (kD)
Intermediate 95 kD
Intermediate 76 kD
Proteolytically cleaved peptide associated with mature 70 kD
WT fibroblasts
Pompefibroblasts
WB: GAA – higher exposure
WB: Actin
15
10075
250150
Proteolytically cleaved peptide associated with mature 70 kD
Intermediate 95 kD
Intermediate 76 kD
WB: GAA – low exposure
WB: GAA – higher exposure
Precursor 110 kD
Precursor 110 kD
Precursor Processed
WT or Pompe patient fibroblasts were incubated with 18.5 nM cipaglucosidase alfa or 500 nM alglucosidase for 16 hours at 37°C. Cells were harvested then lysed and analyzed by Western blotting.
Cipaglucosidase alfa is enriched in bis-phosphorylated N-glycans for improved uptake into cells. Only 18.5 nM cipaglucosidase alfa in the cell culture medium is required to match intracellular GAA levels attained by using 500 nM of the poorly phosphorylated alglucosidase alfa.
20 ng of cipaglucosidase alfa and alglucosidase alfa, as well as lysates of untreated cells were analyzed alongside those of treated cells.
Cipaglucosidase alfa and alglucosidase alfa are processed identically.
Cipaglucosidase alfa is internalized substantially better than the standard of care and is fully processed identical to endogenous
GAA
0 1 2 3 40
20
40
60
80
100
120
Time (Hours)
Nor
mal
ized
GA
A A
ctiv
ity(%
)
Stability in buffer (pH 7.4)
AT-GAA is a two-component investigational therapy comprising of cipaglucosidase alfa and miglustat.
A. Thermal stability of cipaglucosidase alfa alone at neutral (7.4) or acidic (5.2) pH, or in the presence of increasing concentrations of miglustat at pH 7.4. Unfolding of ATB200 was monitored by the increase in the fluorescence of SYPRO Orange as a function of temperature.
B. Time course for ATB200 inactivation (i.e., loss of activity) at 37°C in PBS, pH 7.4 (left) or in human blood ex vivo (right) with and without miglustat.
40 50 60 70 800
20
40
60
80
100
120
Temperature (°C)
Nor
mal
ized
(%)
Syp
ro O
rang
e Fl
uore
scen
ce pH 7.4
pH 7.4 + 10µM miglustat
pH 7.4 + 30µM miglustat
pH 7.4 + 100µM miglustat
pH 5.2
Thermal stability
Stabilizing cipaglucosidase alfa with miglustat – AT-GAA
Miglustat is a small molecule stabilizer of cipaglucosidase alfa
Miglustat improves the protein stability and reduces irreversible inactivation of cipaglucosidase
alfa at neutral/blood pH
Glycogen storage is efficiently reduced by AT-GAA; miglustat has no impact alone
Lysosomal and autophagosomal pathologies are reduced more efficiently by AT-GAA than by miglustat alone
Vehicle Vehicle Alglucosidase alfa Cipaglucosidase alfa + miglustat
Miglustat
Gaa WT Gaa KO
IHC : LAMP1
IHC : LC3 II
Hyperproliferation of lysosomes is a hallmark of PD; LAMP-1 is a lysosomal membrane marker. Lipidated LC3 (LC3 II) is found in the autophagosome membrane and LC3 II positive aggregates in vehicle treated Gaa KO
mice indicate increased autophagy. AT-GAA treatment of Gaa KO mice resulted in substantial reduction in LAMP1 and LC3 II signals in quadriceps compared to
alglucosidase alfa treatment and no reduction was seen in mice treated with miglustat alone. Data shown are representative of images from 7 or 8 animals analyzed per treatment group.
AT-GAA significantly reduces lysosomal build up and autophagy defects in Gaa KO mice
Approximately 12-week-old male Gaa KO mice received two biweekly IV administrations of vehicle, 20 mg/kg alglucosidase alfa, 20 mg/kg cipaglucosidase alfa with miglustat (10 mg/kg miglustat administered orally 30 minutes prior to cipaglucosidase alfa IV injection), or 10 mg/kg miglustat alone (orally).
Tissues were collected 14 days after the second administration, homogenized, and assayed biochemically for glycogen content. Tissue sections were also prepared for IHC (below). Glycogen storage was significantly reduced in quadriceps, heart, and triceps of AT-GAA-treated animals compared to vehicle
treated animals ((* = p<0.05, **=p<0.005, ***=p<0.0005 and ****=p<0.00005) one-way ANOVA with Fisher’s LSD post-hoc test). No reduction was seen in mice treated with miglustat alone. Data are average of measurements from 7 or 8 animals per treatment group and error bars represent SEM.
Co-administration of miglustat with cipaglucosidase alfa improves treatment outcomes but miglustat alone does not impact glycogen reduction in muscles of Gaa KO mice
A. Schematic for periodate oxidation of terminal sialic acid followed by aniline-catalyzed oximation with aminooxyacetic acid (AOAA) introduces anunnatural oxime bond in N-glycans. This could obstruct enzymatic hydrolysis of modified sialic acid by hampering substrate recognition bysialidases/neuraminidases, leading to a block in subsequent glycan processing of modified sialylated complex type N-glycans, as glycandegradation occurs sequentially from the outside-in.
B. RhGAA variants treated with bacterial neuraminidase were subjected to blotting analysis using WGA, which binds to terminal sialic acids.Oxidized rhGAA and rhGAA-AOAA were insensitive to treatment with bacterial neuraminidase in vitro.
0 1 2 3 40
20
40
60
80
100
120
Time (Hours)
Nor
mal
ized
GA
A A
ctiv
ity(%
)
Cipaglucosidase alfa
Cipaglucosidase alfa + 17 µM miglustatCipaglucosidase alfa + 170 µM miglustat
Stability in human blood
A. A stale cell line overexpressing rhGAA was cultured in the presence ofkifunensine to generate rhGAA-kif with mostly high-mannose-type N-glycans.Purified rhGAA-kif was incubated with Endo H for 10 days at 30°C to removehigh mannose type N-glycans while maintaining native structure and activity.LC-MS/MS analysis of rhGAA-kif in parallel with unmodified rhGAA led to thedetection of only high mannose type N-glycans at sites 1-5 but not sites 6 and7, which also had complex glycans. The most abundant glycan structuresobserved at each site
B. Representative Precursor MS1 and HCD MS2 spectra of the glycopeptide, 631ILQFNLLGVPLVGADVCGFLGNTSEE656, bearing the most abundant glycan
0 500 1000 1500 2000 25000.0
0.2
0.4
0.6
0.8
1.0
[4MU-Glc] ( µM)
4MU
rele
ased
(nm
ol /n
M G
AA
/ hr)
0 2000 4000 6000 80000
200
400
600
800
[Glycogen] (µg)
Glu
cose
rele
ased
(µM
/nM
GA
A/ h
r)
rhGAA precursor
rhGAA-AOAA precursor
rhGAA-kif precursor
rhGAA fully processed
rhGAA-AOAA -only proteolytically cleaved
rhGAA-kif + Endo H -only deglycosylated
0
5000
10000
15000
20000
25000
K m ( µ
g)
✱
4MU-Glc kinetics Glycogen kinetics Km glycogen kinetics
A. B.
C. rhGAA
rhGAA + Neuraminidase
D. rhGAA - AOAA
rhGAA - AOAA + Neuraminidase
E. F.
A. B.
are shown. GlcNAc was the only observed structure on sites 1-5, and the most abundant structure on sites 6 and 7 of rhGAA-
structure at Asn 652 (site 5 - in green) in rhGAA-kif rhGAA-kif treated with Endo H are shown. No proteolytic cleavage was observed in Endo H treatment rhGAA-kif . Deglycosylation at site 5 is hypothesized to be necessary for improved access of glycogen to the active site given its proximity to the active site.
A. B. C.
A.
B.
GaaWT
0
100
200
300
400
500
Gly
coge
n(
g/m
gto
talp
rote
in)
Gaa KO
******
****
****
Quadriceps
0
500
1000
Gly
coge
n(
g/m
gto
talp
rote
in)
Gaa KO
GaaWT
********
****
****
Heart
GaaWT
0
200
400
600
Gly
coge
n(
g/m
gto
talp
rote
in)
Gaa KO
******
****
****
**
Triceps
Miglustat
VehicleAlglucosidase alfaCipaglucosidase alfaCipaglucosidase alfa + miglustat
Reference: Xu et al., 2019. JCI Insight.
kif treated with Endo H.