A novel gene editing system to treat both Tay–Sachs and Sandhoff diseases

Abstract

The GM2-gangliosidoses are neurological diseases causing premature death, thus developing effective treatment protocols is urgent. GM2-gangliosidoses result from deficiency of a lysosomal enzyme β-hexosaminidase (Hex) and subsequent accumulation of GM2 gangliosides. Genetic changes in HEXA, encoding the Hex α subunit, or HEXB, encoding the Hex β subunit, causes Tay-Sachs disease and Sandhoff disease, respectively. Previous studies have showed that a modified human Hex μ subunit (HEXM) can treat both Tay-Sachs and Sandhoff diseases by forming a homodimer to degrade GM2 gangliosides. To this end, we applied this HEXM subunit in our PS813 gene editing system to treat neonatal Sandhoff mice. Through AAV delivery of the CRISPR system, a promoterless HEXM cDNA will be integrated into the albumin safe harbor locus, and lysosomal enzyme will be expressed and secreted from edited hepatocytes. Four months after the i.v. of AAV vectors, plasma MUGS and MUG activities reached up to 144-and 17-fold of wildtype levels (n=10, p<0.0001), respectively. More importantly, MUGS and MUG activities in the brain also increased significantly compared with untreated Sandhoff mice (p<0.001). Further, HPLC-MS/MS analysis showed that GM2 gangliosides in multiple tissues, except the brain, of treated mice were reduced to normal levels. Rotarod analysis showed that coordination and motor memory of treated mice were improved (p<0.05). Histological analysis of H&E stained tissues showed reduced cellular vacuolation in the brain and liver of treated Sandhoff mice. These results Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies

“…As a consequence, the knock-in strategy using safe harbors to introduce the cDNA into the genome draws more attention [ 169 , 170 ]. Ou et al, 2020 recently used the albumin locus for the intravenous administration of two AAV8 vectors: one carrying a promoterless HexM cDNA, and other one carrying the Cas9 gene and the gRNA [ 171 ]. This approach allowed the constitutive expression of the HexM under the control of the albumin promoter.…”

“…This increase in the HexM activity correlated with the decrease of the GM2 ganglioside in liver, heart, and spleen. On the other hand, neither a decrease of GM2 ganglioside in the brain nor positive changes on behavioral tests (fear conditioning and pole test) were observed as a result of the treatment [ 171 ]. Despite the above, a significant improvement motor function was observed in treated SD mice compared to untreated animals suggesting a slight therapeutic effect on the CNS.…”

Translating CRISPR-Cas Therapeutics: Approaches and Challenges

“…This was demonstrated in AAV-SaCas9-mediated transgene integration into the albumin safe harbor locus in Sandhoff mice. 180 Sangamo Therapeutics, in a promising preclinical study, used an AAV2/8 zinc finger nuclease editing strategy to insert a-L-iduronidase (IDUA) into the albumin locus, resulting in cognitive improvements in mice with Hurler syndrome (the most severe form of mucopolysaccharidosis [MPS] I). 181 In vivo correction of MPS I has been explored by using CRISPR-Cas to insert the Idua gene in a murine safe harbor locus via HDR.…”

Improvement of motor and behavioral activity in Sandhoff mice transplanted with human CD34+ cells transduced with a HexA/HexB expressing lentiviral vector

“…These include enzyme replacement therapy, allogeneic hematopoietic stem cells (HSCs) and blood transplants, adeno-associated virus delivery of HexA and HexB, substrate reduction therapy, gene editing using a CRISPR system and chaperone therapy. [7][8][9][10][11][12][13][14][15][16][17][18][19] Enzyme replacement therapy is a straight-forward approach of injecting the wild-type (WT) HexA and/or HexB protein product, however, it does not readily enter the central nervous system (CNS). [7][8][9] Allogeneic HSC and blood transplants have been utilized to systemically deliver HexA and HexB to patients through the blood system from donors who are unaffected by TSD or SD.…”

“…Gene therapy and gene editing approaches including adeno-associated virus (AAV) delivery of WT HexA and HexB and the addition of a HexM open reading frame via a CRISPR system are currently being evaluated as viable therapeutic options. [13][14][15]17 AAV has demonstrated strong expression of delivered genes, however, large doses of AAV are required to be injected and do not readily enter the CNS if injected in a systemic manner. Substrate reduction therapy has been evaluated in a group of juvenile GM2 gangliosidosis patients, although it did not display improvement in clinical symptoms.…”