Gene therapies for lysosomal disorders
Inventors
Abeliovich, Asa • Heckman, Laura • RHINN, Herve
Assignees
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Publication Number
US-10837028-B2
Publication Date
2020-11-17
Expiration Date
Abstract
The disclosure relates, in some aspects, to compositions and methods for treatment of diseases associated with aberrant lysosomal function, for example Parkinson's disease and Gaucher disease. In some embodiments, the disclosure provides expression constructs comprising a transgene encoding beta-Glucocerebrosidase (GBA) or a portion thereof, Lysosomal Membrane Protein 2 (LIMP2), Prosaposin, or any combination of the foregoing. In some embodiments, the disclosure provides methods of Parkinson's disease by administering such expression constructs to a subject in need thereof.
Core Innovation
The invention provides recombinant adeno-associated virus (rAAV) vectors for expression of a Gcase protein. The vectors include a promoter operably linked to a transgene insert encoding a Gcase protein, where the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15. The codon optimized version of wild type GBA1 nucleotide sequence eliminates a predicted donor splice site that begins at nucleotide 49 in the wild type GBA1 nucleotide sequence.
In defined embodiments, the rAAV expression cassette includes specific regulatory elements and genome architecture. The document describes an rAAV vector architecture having a 5′ AAV ITR, a CMV enhancer, a CBA promoter, a transgene insert encoding a Gcase protein comprising SEQ ID NO: 15, a WPRE, a Bovine Growth Hormone polyA signal tail, and a 3′ AAV ITR. The document also describes embodiments adding capsid protein specifications and using named regulatory elements including CBA promoter and WPRE.
The problem being solved is lysosomal dysfunction associated with lysosomal disorders, including Parkinson’s disease and Gaucher disease. The vectors are used to increase GCase activity and reduce substrate accumulation of GluSph/GluCer in the context of lysosomal dysfunction. Reported nonclinical results include improvements in motor behavior in mouse models and changes consistent with reduced lysosomal substrate accumulation, along with biodistribution measurements using vector genomes.
Claims Coverage
The provided partial content includes two independent claims, covering (i) a codon-optimized GBA1 (SEQ ID NO: 15) rAAV vector with elimination of a predicted donor splice site, and (ii) a particular rAAV genome cassette order using specific named regulatory elements and flanking AAV ITRs. Across dependent claims, additional inventive features constrain ITR sequences/positions and specify capsid and named cassette components.
Codon optimized GBA1 transgene eliminating a predicted donor splice site
An rAAV vector having a promoter operably linked to a transgene insert encoding a Gcase protein, where the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15 and eliminates a predicted donor splice site that begins at nucleotide 49 in wild type GBA1.
Defined rAAV cassette order with CMV enhancer, CBA promoter, WPRE, and bGH polyA
An rAAV vector with, in 5′ to 3′ order: a 5′ AAV ITR, a CMV enhancer, a CBA promoter, a transgene insert encoding a Gcase protein comprising the nucleotide sequence of SEQ ID NO: 15, a WPRE, a Bovine Growth Hormone polyA signal tail, and a 3′ AAV ITR.
Specified AAV ITR sequence regions
An rAAV vector in which the 5′ ITR nucleic acid sequence corresponds to nucleotides 1–145 of SEQ ID NO: 1 and the 3′ ITR nucleic acid sequence corresponds to nucleotides 3867–4011 of SEQ ID NO: 1.
AAV9 capsid protein
A recombinant AAV characterized in that its AAV capsid protein is the AAV9 capsid protein.
Overall claim coverage centers on rAAV vectors encoding a codon optimized GBA1 (SEQ ID NO: 15) for Gcase expression, with elimination of a predicted donor splice site, and on defined cassette/regulatory element ordering with flanking AAV ITRs. Dependent claim coverage further specifies particular ITR sequence regions and narrows the capsid to AAV9.
Stated Advantages
Increases GCase activity.
Reduces GluSph/GluCer substrate accumulation.
Improves motor behavior in mouse models.
Includes biodistribution measurement using vector genomes.
Includes toxicity evaluation with histopathology/CNS findings reduced at higher rAAV doses.
Documented Applications
Treating Parkinson’s disease (PD) by administering rAAV constructs via CNS delivery (intracisternal/intraparenchymal/intrathecal, including CED) or peripheral routes (e.g., IV) as described in the document.
Nonclinical evaluation in CBE mouse model (conduritol-b-epoxide) and a genetic lysosomal model (4L/PS-NA) to assess GCase activity, lysosomal substrate accumulation, biodistribution, motor behavior, and toxicity.
In vitro expression/transduction in HEK293 cells and a cell-based assay concept for quantifying lysosomal dysfunction and α-Syn pathology.
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