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JP-7855045-B2 - Gene therapy for lysosomal disorders

JP7855045B2JP 7855045 B2JP7855045 B2JP 7855045B2JP-7855045-B2

Inventors

  • アベリオビック，アーサ
  • ヘックマン，ローラ
  • ウォン，リー チン
  • リン，シュアン－ニ
  • ヘフティ，フランツ
  • ライン，エルベ

Assignees

  • プリベイル・セラピューティクス・インコーポレイテッド

Dates

Publication Date
20260507
Application Date
20241009
Priority Date
20190410

Claims (7)

  1. A method for quantifying progranulin (PGRN) protein levels in cerebrospinal fluid (CSF) samples, (a) Diluting the CSF sample in a master mixture containing dithiothreitol (DTT) and sample buffer, (b) Filling the wells of the capillary cartridge with the diluted CSF sample, anti-progranulin antibody, secondary antibody for detecting the anti-progranulin antibody, luminol, and peroxide, (c) Loading the capillary cartridge into an automated Western blot immunoassay instrument, (d) Using the automated Western blot immunoassay instrument, calculate the signal intensity, peak area, and signal-to-noise ratio, (e) The progranulin protein level in the CSF sample is quantified as the peak area for the immunoreactive anti-progranulin antibody, The aforementioned CSF sample (i) In order from 5' to 3', (a) Adeno-associated virus 2 (AAV2) ITR, (b) Cytomegalovirus (CMV) enhancer, (c) Chicken beta-actin (CBA) promoter, (d) A transgene insertion encoding the progranulin (PGRN) protein, wherein the transgene insertion contains the nucleotide sequence of Sequence ID No. 68, (e) Woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE), (f) Bovine growth hormone polyA signaling tail, and (g) AAV2 ITR A recombinant adeno-associated virus (rAAV) vector comprising nucleic acids containing an expression construct, (ii) Adeno-associated virus (AAV) capsid protein, The method is based on subjects who have been previously administered recombinant adeno-associated virus (rAAV) containing the above.
  2. The method according to claim 1, wherein the CSF sample is of human origin.
  3. The method according to claim 1, wherein the CSF sample is from a subject having or suspected of having frontotemporal dementia (FTD).
  4. The method according to claim 1, wherein the FTD is an FTD having a GRN mutation.
  5. The method according to claim 1, wherein the CSF sample is derived from a primate other than a human.
  6. The method according to claim 1 , wherein the AAV capsid protein is the AAV9 capsid protein.
  7. The method according to any one of claims 1 to 6 , wherein the dilution step is a four-fold dilution.

Description

Cross-reference of Related Applications This application claims priority to U.S. Provisional Patent Application No. 62/988,665 filed March 12, 2020, U.S. Provisional Patent Application No. 62/960,471 filed January 13, 2020, U.S. Provisional Patent Application No. 62/954,089 filed December 27, 2019, U.S. Provisional Patent Application No. 62/934,450 filed November 12, 2019, and U.S. Provisional Patent Application No. 62/831,846 filed April 10, 2019. The disclosures of each of these applications are incorporated herein by reference in their entirety. Description of electronically submitted text files: The contents of text files electronically submitted with this specification are incorporated herein by reference in their entirety: a computer-readable formatted copy of the sequence listing (file name: PRVL_010_05WO_SeqList.txt, date recorded: April 10, 2020, file size approximately 612,902 bytes). Field: This disclosure relates to the field of gene therapy and methods of using it. Gaucher disease is a rare congenital anomaly of sphingoglycolipid metabolism due to a deficiency of lysosomal acidic β-glucocerebrosidase (Gcase, "GBA"). Patients suffer from hepatosplenomegaly, bone marrow failure leading to pancytopenia, pulmonary impairment and fibrosis, and non-CNS symptoms and findings including bone defects. Furthermore, a significant number of patients suffer from neurological symptoms including saccadic eye movements and gaze defects, seizures, cognitive impairment, developmental delay, and motor disorders including Parkinson's disease. Several treatments exist to address peripheral diseases in the hematopoietic bone marrow and viscera, as well as the main clinical manifestations. These include enzyme replacement therapy, chaperone-like small molecule drugs that bind to the deficient Gcase to improve its stability, and substrate reduction therapy that blocks the production of substrates that accumulate in Gaucher disease and lead to symptoms and findings. However, other aspects of Gaucher disease (particularly those affecting the skeleton and brain) appear to be refractory to treatment. Progranulin (PGRN) is an additional protein associated with lysosomal function. PGRN is encoded by the GRN gene. In humans, GRN haploy failure increases the risk of developing FTD-GRN (frontotemporal dementia with GRN mutations), a neurodegenerative disease characterized by executive function impairment, behavioral changes, and language disorders, accompanied by atrophy of the frontal and temporal lobes, to approximately 90%. There are no disease-modifying therapies for FTD patients. This specification provides a method for treating subjects having or suspected of having frontotemporal dementia with a GRN mutation, the method comprising administering to the subject a recombinant adeno-associated virus (rAAV) comprising: (i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a PGRN protein, the transgene insert comprising the nucleotide sequence of SEQ ID NO: 68, and (ii) an AAV9 capsid protein. In some embodiments, the rAAV is administered to the subject in doses ranging from about 1 × 10¹³ vector genome (vg) to about 7 × 10¹⁴ vg. In some embodiments, the rAAV is administered by injection into the cisterna magna. In some embodiments, the promoter operably linked to the transgene insert encoding the PGRN protein is a chicken beta-actin (CBA) promoter. In some embodiments, the rAAV vector further comprises a cytomegalovirus (CMV) enhancer. In some embodiments, the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulator (WPRE). In some embodiments, the rAAV vector further comprises a bovine growth hormone polyA signaling tail. In some embodiments, the nucleic acid comprises two adeno-associated virus inverted terminal repeat (ITR) sequences adjacent to the expression construct. In some embodiments, each ITR sequence is a wild-type AAV2 ITR sequence. In some embodiments, the rAAV vector further comprises a TRY region between the 5' ITR and the expression construct, the TRY region comprising SEQ ID NO: 28. This specification provides a method for treating subjects having or suspected to have frontotemporal dementia with a GRN mutation, the method comprising administering to the subject an rAAV comprising: (i) a nucleic acid-containing rAAV vector comprising, in order from 5' to 3', (a) an AAV2 ITR, (b) a CMV enhancer, (c) a CBA promoter, (d) a transgene insert encoding the PGRN protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68, (e) WPRE, (f) a bovine growth hormone poly-A signaling tail, and (g) an AAV2 ITR, and (ii) an AAV9 capsid protein. In some embodiments, the rAAV is administered to the subject in doses ranging from about 1 × 10¹³ vg to about 7 × 10¹⁴ vg. In some embodiments, the rAAV is administered by injection into the cisterna magna. In some embodiments, rAAV