Dual Purpose Vectors for Rare Neurological Diseases
Authors: Brian W. Bigger
Journal: Molecular Therapy (2020)

Gene therapy offers much hope for debilitating genetic disorders such as lysosomal storage diseases, which often exhibit a severe phenotype that includes neurological complications. The fact that they tend to be single gene defects and that supply of the missing enzymes through the bloodstream results in cellular uptake and translocation into lysosomes makes them amenable to gene therapy. Sandhoff and Tay-Sachs diseases, however, have additional complications that make treatment particularly challenging. A new study by Lahey et al.,1Lahey H.G. Hwang M. et al.Pronounced therapeutic benefit of a single bidirectional AAV vector administered systemically in Sandhoff mice.Mol Ther. 2020; 28 (this issue): 2150-2160Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar published in this issue of Molecular Therapy, constitutes a major advance toward treatment of these devastating GM2 gangliosidoses that cause extensive damage to neurons in the brain. Sandhoff disease is caused by deficiencies in the HEXB gene, leading to accumulation of undegraded GM2 gangliosides, and is clinically indistinguishable from its more well-known counterpart, Tay-Sachs disease, caused by HEXA mutations. HEXA and HEXB genes encode, respectively, α and β subunits of the hexosaminidase enzyme. GM2 ganglioside breakdown requires two isoforms of the hexosaminidase enzyme (confusingly named Hex-A and Hex-B), which are made up of, respectively, (α-β) and (β-β) subunits (Figure 1). Hex-A also requires a co-factor (GM2 activator protein) for activity. Delivery of the β subunit alone as a gene therapy in the Sandhoff mouse model appears to lead to a skewing of enzyme assembly toward Hex-B, with limitation of α subunit availability, perhaps due to steric effects arising from over-production of the Hex-B enzyme2Cachón-González M.B. Wang S.Z. McNair R. Bradley J. Lunn D. Ziegler R. Cheng S.H. Cox T.M. Gene transfer corrects acute GM2 gangliosidosis--potential therapeutic contribution of perivascular enzyme flow.20. 2012: 1489-1500Google Scholar (Figure 1). Thus, for successful enzyme assembly of both Hex-A and Hex-B enzymes, provision of both HEXA and HEXB genes in roughly equal dosage is critical to successful Sandhoff and Tay-Sachs therapies. Previous efforts to correct Sandhoff or Tay-Sachs have attempted to deliver two adeno-associated viral (AAV) vectors expressing HEXA and HEXB, respectively,2Cachón-González M.B. Wang S.Z. McNair R. Bradley J. Lunn D. Ziegler R. Cheng S.H. Cox T.M. Gene transfer corrects acute GM2 gangliosidosis--potential therapeutic contribution of perivascular enzyme flow.20. 2012: 1489-1500Google Scholar,3Sargent T.J. Wang S. Bradley J. Smith N.J.C. Raha A.A. McNair R. Ziegler R.J. Cheng S.H. Cox T.M. Cachón-González M.B. Adeno-associated virus-mediated expression of β-hexosaminidase prevents neuronal loss in the Sandhoff mouse brain.20. 2011: 4371-4380Google Scholar which leads to multiple problems. These include reliance on efficient co-delivery of both vectors to the same cell, which is particularly difficult when targeting the brain by direct injection and even more challenging with an intravenous delivery approach. An alternative strategy was to develop a HEXA modified gene, incorporating sections of the similar HEXB, that could form a monomeric dimer and bind GM2 activator protein (dubbed Hex-M).4Karumuthil-Melethil, S., Kalburgi, S.N., Thompson, P., Tropak, M., Kaytor, M.D., Keimel, J.G., Mark, B.L., Mahuran, D., Walia, J.S., and Gray, S.J. (2016). 27, 509–521.Google Scholar While this may prove quite successful for Tay-Sachs, it still does not solve the problem of Sandhoff. Lahey et al.1Lahey H.G. Hwang M. et al.Pronounced therapeutic benefit of a single bidirectional AAV vector administered systemically in Sandhoff mice.Mol Ther. 2020; 28 (this issue): 2150-2160Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar have neatly solved this old problem by developing a single AAV vector expressing both HEXA and HEXB genes from a bidirectional promoter. They demonstrate that these constructs are able to achieve long-term expression, reduce GM2 ganglioside accumulation, and improve behavioral outcomes and survival in Sandhoff disease mice, with the AAV9-Bic construct (a vector containing a bidirectional CBA promoter—a cytomegalovirus [CMV] enhancer, flanked by two β-actin promoters) demonstrating the highest level of correction. This result is truly impressive, given that each gene is approximately 1.6 Kb in size, leaving precious little effective packaging capacity to work with when incorporated into an AAV vector. Both transcripts were able to effectively deliver each Hex subunit and correct disease in the mouse model. Importantly, gene expression was sustained for up to 2 years in some cases, suggesting that promoter methylation, typically a feature of unmodified CMV promoters, may not be a significant factor in this design. Innovative promoter design in gene therapy vectors has perhaps not received as much attention in recent years as it deserves. In order to improve the effectiveness, specificity, and safety of vectors, optimal design of promoters remains critical. Bidirectional promoters, such as the one described here, also open up the prospect for designing effective and controlled regulated gene expression. Many such systems require dual gene expression, although few are sufficiently tightly regulated to be suitable for clinical use. Lahey et al.1Lahey H.G. Hwang M. et al.Pronounced therapeutic benefit of a single bidirectional AAV vector administered systemically in Sandhoff mice.Mol Ther. 2020; 28 (this issue): 2150-2160Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar have elegantly addressed an old problem in the lysosomal disease community with a vector that should correct both Sandhoff and Tay-Sachs disease, and its design likely has even broader applicability. Going forward, the same research group is in the unique position to test the concept in a sheep model of Tay-Sachs disease (because mice possess an alternative pathway for GM2 ganglioside catabolism, murine models of Tay-Sachs disease are not suitable for development of therapies). Experiments in these large animals should prove informative if this vector does indeed serve the dual purpose of treating both Sandhoff and Tay-Sachs. Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff MiceLahey et al.Molecular TherapyJune 19, 2020In BriefTay-Sachs and Sandhoff diseases are caused by mutations in the HEXA and HEXB genes, respectively, which impact the activity of the heterodimeric lysosomal enzyme β-hexosaminidase A, and result in the accumulation of GM2 ganglioside in CNS. HexA is the only isozyme in human that hydrolyzes GM2 ganglioside, and co-expression of both proteins is necessary to produce high enzyme levels for cross-correction. An AAV9 vector expressing both proteins from a central promoter was administered systemically in Sandhoff mice and shown to reduce GM2 ganglioside levels, improve behavioral performance, and extend median survival to 634 days. Full-Text PDF Open Archive