Phosphorylation is the process of adding a phosphate group to a target molecule. For many years, research into treatments for lysosomal storage disorder (LSD) focused on finding a way to add a phosphate group to the sixth location on a target mannose molecule to create mannose 6-phosphate (M6P).
The following diagram(1) depicts the steps necessary to complete this process.
GlcNAc-1-phosphotransferase is a naturally occurring enzyme that resides in the Golgi apparatus within cells. GlcNAc-1-phosphotransferase catalyzes the transfer of GlcNAc-1-phosphate from uridine diphosphate (UDP) onto certain terminal mannose residues of the N-linked oligosaccharides on enzymes destined for the lysosome.
N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (NAGPA), also known as uncovering enzyme, removes the covering GlcNAc group, thereby exposing mannose 6-phosphate.
S1-S3 variant of GlcNAc-1-phosphotransferase
Although scientists have understood the process for phosphorylating lysosomal enzymes for some time, no one has been able to control GlcNAc-1-phosphotransferase to enhance the level of M6P on enzyme replacement therapy (ERT) enzymes. The problem is that GlcNAc-1-phosphotransferase is difficult to control in laboratory conditions and manufacturing processes.
In 2017, Stuart Kornfeld and Lin Liu achieved a breakthrough when they created a controllable variant of GlcNAc-1-phosphotransferase — the S1-S3 variant. When cells were given both the DNA sequences for the S1-S3 variant and various lysosomal enzymes, the cells were able to produce the lysosomal enzymes with high M6P content. Drs. Kornfeld and Liu published their results in a leading peer-reviewed journal(2), clearly showing that the S1-S3 variant can enhance the M6P content of lysosomal enzymes and that the resulting lysosomal enzymes are better able to bind to cell surface receptors.
* p<.05; ** p<.01; LAMAN: lysosomal acid alpha-mannosidase for alpha-mannosidosis. GAA: acid alpha-glucosidase for Pompe disease. GLA: alpha-galactosidase A for Fabry disease. GBA: beta-glucocerebrosidase for Gaucher disease.
Bicistronic vector platform
With the S1-S3 variant technology, we can co-express separate DNA sequences for the S1-S3 variant and the lysosomal enzyme in the same cell to create M6P-rich lysosomal enzymes. While this is a significant advancement, it has two shortfalls that we have been working to address:
1. Since separate DNA sequences need to be put into cells, a great deal of experimentation needs to be done for each additional lysosomal enzyme. We essentially need to redo most of the work for each additional lysosomal enzyme we want to create.
2. Since separate DNA sequences are involved, the challenge of putting both into a virus to create a gene therapy is almost unsurmountable.
Consequently, our goal was to create a general vehicle whereby the S1-S3 variant of GlcNAc-1-phosphotransferase would be expressed along with any therapeutic lysosomal enzyme. As an analogy, we wanted to build a pickup truck with a payload bed that could hold any enzyme, so the resulting fully loaded truck leads to an M6P-rich enzyme. Scientifically speaking, we aimed to create a single DNA expression vector that would simultaneously express two separate genes. Such a vector, with two DNA sequences, or cistrons, is known as a bicistronic vector.
Our molecular biologists have done some phenomenal work and have created this bicistronic platform. Using this platform, we have already created six lysosomal enzymes rich in M6P content.
1. Kang et al. Mutations in the Lysosomal Enzyme – Targeting Pathway and Persistent Stuttering. N Engl J Med 2010;362:677-685.
2. Lin Liu, Wang-Sik Lee, Balraj Doray, and Stuart Kornfeld. Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy,
Mol Ther Methods Clin Dev. 2017 Jun 16; 5: 59–65.