Expansion of B4GALT7 linkeropathy phenotype to include perinatal lethal skeletal dysplasia.

Proteoglycans have a core polypeptide connected to glycosaminoglycans (GAGs) via a common tetrasaccharide linker region. Defects in enzymes that synthesize the linker result in a group of autosomal recessive conditions called "linkeropathies". Disease manifests with skeletal and connective tissue features, including short stature, hyperextensible skin, and joint hypermobility. We report a family with three affected pregnancies showing short limbs, cystic hygroma, and perinatal death. Two spontaneously aborted; one survived 1 day after term delivery, and had short limbs, bell-shaped thorax, 11 ribs, absent thumbs, and cleft palate. Exome sequencing of the proband and one affected fetus identified compound heterozygous missense variants, NM_007255.3: c.808C>T (p.(Arg270Cys)) and NM_007255.3: c.398A>G (p.(Gln133Arg)), in B4GALT7, a gene required for GAG linker biosynthesis. Homozygosity for p.(Arg270Cys), associated with partial loss of B4GALT7 function, causes Larsen of Reunion Island syndrome (LRS), however no previous studies have linked p.(Gln133Arg) to disease. The p.(Gln133Arg) and p.(Arg270Cys) variants were transfected into CHO pgsB-618 cells. High protein expression of p.(Gln133Arg) was found, with mislocalization, compared to p.(Arg270Cys) that had a normal Golgi-like pattern. The p.(Gln133Arg) had almost no enzyme activity and little production of heparan sulfate GAGs, while p.(Arg270Cys) only had 17% of wild-type activity. These findings expand the phenotype of B4GALT7-related linkeropathies to include lethal skeletal dysplasia due to more severe loss of function.

An Isolated Limb Infusion Method Allows for Broad Distribution of rAAVrh74.MCK.GALGT2 to Leg Skeletal Muscles in the Rhesus Macaque.

Recombinant adeno-associated virus (rAAV)rh74.MCK.GALGT2 is a muscle-specific gene therapy that is being developed to treat forms of muscular dystrophy. Here we report on an isolated limb infusion technique in a non-human primate model, where hindlimb blood flow is transiently isolated using balloon catheters to concentrate vector in targeted leg muscles. A bilateral dose of 2.5 × 1013 vector genomes (vg)/kg/limb was sufficient to induce GALGT2-induced glycosylation in 10%-60% of skeletal myofibers in all leg muscles examined. There was a 19-fold ± 6-fold average limb-wide increase in vector genomes per microgram genomic DNA at a bilateral dose of 2.5 × 1013 vg/kg/limb compared with a bilateral dose of 6 × 1012 vg/kg/limb. A unilateral dose of 6 × 1013 vg/kg/limb showed a 12- ± 3-fold increase in treated limb muscles compared to contralateral untreated limb muscles, which received vector only after release into the systemic circulation from the treated limb. Variability in AAV biodistribution between different segments of the same muscle was 125% ± 18% for any given dose, while variability between the same muscle for any given treatment dose was 45% ± 7%. These experiments demonstrate that treatment of muscles throughout the leg with rAAVrh74.MCK.GALGT2 can be accomplished safely using an isolated limb infusion technique, where balloon catheters transiently isolate the limb vasculature, but that intra- and inter-muscle transduction variability is a significant issue.