Gene therapies for lysosomal disorders
Inventors
Abeliovich, Asa • Heckman, Laura • RHINN, Herve
Assignees
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Publication Number
US-11802294-B2
Publication Date
2023-10-31
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 relates to gene-therapy concepts for lysosomal disorders, including Gaucher disease and Lewy body dementia, using adeno-associated virus (AAV) expression constructs. The constructs encode a beta-Glucocerebrosidase (Gcase) protein coding sequence together with a coding sequence encoding an inhibitory nucleic acid targeting α-Synuclein. The disclosed expression constructs are flanked by AAV inverted terminal repeats (ITR) sequences, including variants involving a modified “D” region and an optional “TRY region.”
The document describes rAAV vector design choices such as regulatory and sequence elements, including promoters such as CBA/CAG, and posttranscriptional regulatory elements such as WPRE, and polyadenylation elements. The approach includes the use of a transgene containing the Gcase protein coding sequence (SEQ ID NO: 15) and the inhibitory nucleic acid targeting α-Synuclein (SEQ ID NO: 20), with a vector design rationale for the modified AAV ITR “D” region placement to support efficient packaging and cell transduction and to address potential toxicity reduction.
The disclosure further frames delivery concepts aimed at the central nervous system (CNS), including direct brain/spinal cord injection routes and peripheral administration options, and also includes convection enhanced delivery (CED) concepts for CNS delivery. The document includes example frameworks and study structures and reports preclinical/ex vivo/in vitro results, including that GBA1 rAAV increases GCase activity and reduces glucosylceramide/glucosylsphingosine substrates, alongside biodistribution and toxicity observations.
Claims Coverage
The independent claims cover six main inventive feature groupings: an isolated AAV-flanked dual-transgene nucleic acid; an rAAV vector with defined regulatory and ITR elements; an rAAV particle composition; an alternative Baculovirus vector carrying the same sequence payload; a cell system with separate rep/cap and payload vectors; and an explicit CNS treatment indication in the context of rAAV administration.
AAV-flanked isolated nucleic acid with dual transgene payload
An isolated nucleic acid comprising an expression construct with a beta-Glucocerebrosidase (Gcase) protein coding sequence comprising the sequence set forth in SEQ ID NO: 15 and a coding sequence encoding an inhibitory nucleic acid targeting α-Synuclein, wherein the inhibitory nucleic acid is encoded by the sequence set forth in SEQ ID NO: 20, wherein two AAV inverted terminal repeats (ITR) sequences flank the expression construct.
rAAV vector with Gcase and α-Synuclein inhibitory nucleic acid expression construct
A recombinant adeno-associated virus (rAAV) vector comprising an expression construct comprising a transgene with a Gcase protein coding sequence comprising the sequence set forth in SEQ ID NO: 15 and a coding sequence encoding an inhibitory nucleic acid targeting α-Synuclein, wherein the inhibitory nucleic acid is encoded by the sequence set forth in SEQ ID NO: 20, wherein the expression construct is flanked by two AAV ITR sequences.
rAAV vector defined by regulatory elements and terminal elements in 5’ to 3’ order
An rAAV vector comprising a nucleic acid comprising, in 5’ to 3’ order: a 5’ AAV ITR, a CMV enhancer, a CBA promoter, a transgene comprising a Gcase protein coding sequence comprising the sequence set forth in SEQ ID NO: 15 and a coding sequence encoding an inhibitory nucleic acid targeting α-Synuclein encoded by the sequence set forth in SEQ ID NO: 20, a WPRE, a Bovine Growth Hormone poly A signal tail, and a 3’ AAV ITR.
rAAV particle comprising AAV capsid protein and the defined rAAV vector
A recombinant AAV (rAAV) particle comprising an AAV capsid protein and an rAAV vector as recited in the preceding vector definition.
Baculovirus vector containing the defined Gcase and α-Synuclein inhibitory nucleic acid sequences
A Baculovirus vector comprising (i) the sequence set forth in SEQ ID NO: 15 and (ii) a coding sequence encoding an inhibitory nucleic acid targeting α-Synuclein, wherein the inhibitory nucleic acid is encoded by the sequence set forth in SEQ ID NO: 20.
Cell with separate AAV rep/cap vector and payload expression construct vector
A cell comprising: (i) a first vector encoding one or more AAV rep proteins and/or one or more AAV cap proteins; and (ii) a second vector comprising an expression construct comprising a Gcase protein coding sequence comprising the sequence set forth in SEQ ID NO: 15 and a coding sequence encoding an inhibitory nucleic acid targeting α-Synuclein, wherein the inhibitory nucleic acid is encoded by the sequence set forth in SEQ ID NO: 20.
Together, the independent claims define a shared genetic payload consisting of a Gcase coding sequence (SEQ ID NO: 15) combined with an inhibitory nucleic acid targeting α-Synuclein (SEQ ID NO: 20). Coverage includes isolated AAV-flanked nucleic acids, rAAV vectors with defined regulatory and terminal components, rAAV particle compositions including an AAV capsid protein, an alternative Baculovirus vector option carrying the same sequence payload, and a cell configuration using separate vectors for AAV rep/cap and for the expression construct.
Stated Advantages
GBA1 rAAV increases GCase activity.
GBA1 rAAV reduces glucosylceramide/glucosylsphingosine substrates.
Vector design of the modified AAV ITR “D” region placement supports efficient packaging and cell transduction.
Modified AAV ITR “D” region placement is described as potentially reducing toxicity.
Documented Applications
Treating a subject with Lewy Body Dementia associated with a GBA1 mutation by administering the rAAV via direct injection into the central nervous system (CNS).
CNS-directed delivery approaches including direct brain/spinal cord injection routes, and convection enhanced delivery (CED) concepts for CNS delivery.
Example preclinical/ex vivo/in vitro study frameworks including measurements of GCase activity, reductions in glucosylceramide/glucosylsphingosine substrates, and biodistribution and toxicity observations [procedural detail omitted for safety].
Applications framed for lysosomal disorders including Gaucher disease and Lewy body dementia.
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