EP4713028A1 - Formulations comprenant un aav recombinant et leurs procédés d'administration - Google Patents

Formulations comprenant un aav recombinant et leurs procédés d'administration

Info

Publication number
EP4713028A1
EP4713028A1 EP24734419.5A EP24734419A EP4713028A1 EP 4713028 A1 EP4713028 A1 EP 4713028A1 EP 24734419 A EP24734419 A EP 24734419A EP 4713028 A1 EP4713028 A1 EP 4713028A1
Authority
EP
European Patent Office
Prior art keywords
pharmaceutical formulation
concentration
raav
endotoxin
formulation comprises
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24734419.5A
Other languages
German (de)
English (en)
Inventor
Heather LAU
Julie Yu Wei
Kirin JAMISON
Jan PANTELI
Sharyl Lynne Fyffe-Maricich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ultragenyx Pharmaceutical Inc
Original Assignee
Ultragenyx Pharmaceutical Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ultragenyx Pharmaceutical Inc filed Critical Ultragenyx Pharmaceutical Inc
Publication of EP4713028A1 publication Critical patent/EP4713028A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present disclosure relates generally to formulations comprising recombinant adeno- associated virus (rAAV) and methods of their use in gene therapy for the treatment of various genetic disorders.
  • rAAV recombinant adeno- associated virus
  • AAV adeno-associated virus
  • CDKL5 deficiency disorder a rare neurodevelopmental disease caused by mutations in the CDKL5 gene which can manifest in a broad range of clinical symptoms and severity.
  • Hallmarks of CDD include infantile-onset refractory epilepsy, developmental delay, intellectual disability, visual impairment, lack of speech, hypotonia, motor dysfunction, sleep disturbances, gastrointestinal dysfunction, and breathing difficulties. Although rare, the occurrence is believed to be approximately 1 in 40,000 - 60,000 live births, making it one of the most common forms of genetic epilepsy.
  • AAV for the treatment of CDD and formulations comprising the same are described in WO/2021/163322 and WO/2023/023590, respectively, the disclosures of which are herein incorporated by reference in their entireties.
  • the present disclosure provides methods for preparing pharmaceutical formulations comprising rAAV suitable for administration to the CNS via intrathecal (IT), intracerebroventricular (ICV), and/or intracisternal magna (ICM) routes. Also described herein are pharmaceutical formulations comprising rAAV suitable for administration to the CNS and methods of their use in the treatment of CNS disorders, e.g., rare genetic CNS disorders such as CDD.
  • the present disclosure provides pharmaceutical formulations comprising recombinant adeno-associated virus (rAAV) and methods of their use in gene therapy. Also provided herein are methods for preparing pharmaceutical formulations comprising rAAV suitable for administration to the central nervous system (CNS) via intrathecal (IT), intracerebroventricular (ICV), and/or intracisternal magna (ICM) routes.
  • rAAV recombinant adeno-associated virus
  • ICM intracisternal magna
  • the present disclosure provides a pharmaceutical formulation for administration to the CNS of a subject, the pharmaceutical formulation comprising a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 13 genome copies (GC)/mL and having an endotoxin concentration of less than or equal to 0.2 endotoxin units (EU)/mL.
  • rAAV recombinant adeno-associated virus
  • the present disclosure provides methods of treating a central nervous system (CNS) disorder in a subject comprising administering to the CNS of the subject a pharmaceutical formulation comprising a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.2 EU/mL.
  • a pharmaceutical formulation comprising a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.2 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises an rAAV at a concentration of at least 5 x 10 13 GC/mL, at least 6 x 10 13 GC/mL, at least 7 x IO 13 GC/mL, at least 8 x IO 13 GC/mL, at least 9 x IO 13 GC/mL, at least 1 x 10 14 GC/mL, at least 1.5 x 10 14 GC/mL, at least 2 x 10 14 GC/mL, at least 3 x 10 14 GC/mL, at least 4 x 10 14 GC/mL, at least 5 x 10 14 GC/mL, at least 6 x 10 14 GC/mL, at least 7 x 10 14 GC/mL, at least 8 x 10 14 GC/mL, at least 9 x 10 14 GC/mL, at least 1 x IO 15 GC/mL, at least 3 x IO 15 GC/mL, or at
  • the pharmaceutical formulation comprises an rAAV at a concentration of at least 5 x 10 13 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 6 x 10 13 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 7 x 10 13 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 8 x 10 13 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 9 x 10 13 GC/mL.
  • the pharmaceutical formulation comprises an rAAV at a concentration of at least 1 x 10 14 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 1.5 x 10 14 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 2 x 10 14 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 3 x 10 14 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 4 x 10 14 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 5 x 10 14 GC/mL.
  • the pharmaceutical formulation comprises an rAAV at a concentration of at least 6 x 10 14 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 7 x 10 14 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 8 x 10 14 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 9 x 10 14 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 1 x 10 15 GC/mL. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of at least 3 x 10 15 GC/mL.
  • the pharmaceutical formulation comprises an rAAV at a concentration of at least 5 x 10 15 GC/mL.
  • the pharmaceutical formulation comprises an rAAV at a concentration of 1 x 10 14 GC/ml to 3 x 10 15 GC/ml (e.g., 1 x 10 14 to 2 x 10 14 GC/ml, 2 x 10 14 to 3 x 10 14 GC/ml, 3 x 10 14 to 4 x 10 14 GC/ml, 4 x 10 14 to 5 x 10 14 GC/ml, 5 x 10 14 to 6 x 10 14 GC/ml, 6 x 10 14 to 7 x 10 14 GC/ml, 7 x 10 14 to 8 x 10 14 GC/ml, 8 x 10 14 to 9 x 10 14 GC/ml, 9 x 10 14 to 1 x 10 15 GC/ml, 1 x 10 15 to 2 x 10 15 GC/ml
  • the pharmaceutical formulation comprises an rAAV at a concentration of 1 x 10 14 to 2 x 10 14 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 2 x 10 14 to 3 x 10 14 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 3 x 10 14 to 4 x 10 14 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 4 x 10 14 to 5 x 10 14 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 5 x 10 14 to 6 x 10 14 GC/ml.
  • the pharmaceutical formulation comprises an rAAV at a concentration of 6 x 10 14 to 7 x 10 14 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 7 x 10 14 to 8 x 10 14 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 8 x 10 14 to 9 x 10 14 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 9 x 10 14 to 1 x 10 15 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 1 x 10 15 to 2 x 10 15 GC/ml.
  • the pharmaceutical formulation comprises an rAAV at a concentration of 2 x 10 15 to 3 x 10 15 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 2 x 10 14 to 2 x 10 15 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 3 x 10 14 to 1 x 10 15 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 4 x 10 14 to 9 x 10 14 GC/ml. In some embodiments, the pharmaceutical formulation comprises an rAAV at a concentration of 5 x 10 14 to 8 x 10 14 GC/ml.
  • the pharmaceutical formulation has an endotoxin concentration of less than or equal to 0.2 EU/mL, less than or equal to 0.15 EU/mL, less than or equal to 0.1 EU/mL, less than or equal to 0.05 EU/mL, less than or equal to 0.03 EU/mL, or less than or equal to 0.01 EU/mL. In some embodiments, the pharmaceutical formulation has an endotoxin concentration of less than or equal to 0.2 EU/mL. In some embodiments, the pharmaceutical formulation has an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation has an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • the pharmaceutical formulation has an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation has an endotoxin concentration of less than or equal to 0.03 EU/mL. In some embodiments, the pharmaceutical formulation has an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 6 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 6 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 6 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 6 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 6 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 7 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 7 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 7 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 7 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 7 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 8 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 8 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 8 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 8 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 8 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 9 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.2 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 9 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 9 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 9 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 9 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 9 x 10 13 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 10 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.2 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 10 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 10 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 10 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 10 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 10 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 1.5 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 1.5 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 1.5 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 2 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 2 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 2 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 2 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 2 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 3 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 3 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 3 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 3 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 3 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 4 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 4 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 4 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 4 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 4 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 6 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 6 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 6 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 6 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 6 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 7 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 7 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 7 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 7 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 7 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 8 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 8 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 8 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 8 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 8 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 9 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 9 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 9 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 9 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 9 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 1 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.2 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 1 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 1 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 1 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 1 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 1 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 3 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.2 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 3 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 3 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 3 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 3 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 3 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.2 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.15 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.05 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.03 EU/mL. In some embodiments, the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 5 x 10 15 GC/mL and having an endotoxin concentration of less than or equal to 0.01 EU/mL.
  • rAAV recombinant adeno-associated virus
  • the pharmaceutical formulation comprises an rAAV at a concentration of 1 x 10 14 GC/ml in the pharmaceutical formulation. In one embodiment according to the above aspects, the pharmaceutical formulation comprises an rAAV at a concentration of 1.5 x 10 14 GC/ml in the pharmaceutical formulation. In one embodiment according to the above aspects, the pharmaceutical formulation comprises an rAAV at a concentration of 2 x 10 14 GC/ml in the pharmaceutical formulation.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 1 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno-associated virus (rAAV) at a concentration of at least 1.5 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.1 EU/mL.
  • the pharmaceutical formulation comprises a recombinant adeno- associated virus (rAAV) at a concentration of at least 2 x 10 14 GC/mL and having an endotoxin concentration of less than or equal to 0.2 EU/mL.
  • rAAV recombinant adeno- associated virus
  • the pharmaceutical formulation comprises a total rAAV dose of 3.6 x 10 14 GC to 3 x 10 15 GC (e.g., 3.6 x 10 14 to 4 x 10 14 GC, 4 x 10 14 to 5 x 10 14 GC, 5 x 10 14 to 6 x 10 14 GC, 6 x 10 14 to 7 x 10 14 GC, 7 x 10 14 to 8 x
  • the pharmaceutical formulation comprises a total rAAV dose of 3.6 x 10 14 to 4 x 10 14 GC. In some embodiments, the pharmaceutical formulation comprises a total rAAV dose of 4 x 10 14 to 5 x 10 14 GC. In some embodiments, the pharmaceutical formulation comprises a total rAAV dose of 5 x 10 14 to 6 x 10 14 GC.
  • the pharmaceutical formulation comprises a total rAAV dose of 6 x 10 14 to 7 x 10 14 GC. In some embodiments, the pharmaceutical formulation comprises a total rAAV dose of 7 x 10 14 to 8 x 10 14 GC. In some embodiments, the pharmaceutical formulation comprises a total rAAV dose of 8 x 10 14 to 9 x 10 14 GC. In some embodiments, the pharmaceutical formulation comprises a total rAAV dose of 9 x 10 14 to 1 x 10 15 GC. In some embodiments, the pharmaceutical formulation comprises a total rAAV dose of 1 x 10 15 to 2 x 10 15 GC. In some embodiments, the pharmaceutical formulation comprises a total rAAV dose of 2 x 10 15 to 3 x 10 15 GC.
  • the pharmaceutical formulation comprises a total rAAV dose of 4 x IO 15 to 2 x 10 15 GC. In some embodiments, the pharmaceutical formulation comprises a total rAAV dose of 5 x 10 14 to 1 x 10 15 GC. In some embodiments, the pharmaceutical formulation comprises a total rAAV dose of 6 x 10 14 to 9 x 10 14 GC.
  • the pharmaceutical formulation is dosed at 0.1 x 10 12 GC/g brain to 3 x 10 12 GC/g brain (e.g., 0.1 x 10 12 to 0.5 x 10 12 GC/g brain, 0.5 x 10 12 to 1 x 10 12 GC/g brain, 1 x 10 12 to 1.5 x 10 12 GC/g brain, 1.5 x 10 12 to 2 x 10 12 GC/g brain, 2 x 10 12 to 2.5 x 10 12 GC/g brain, 2.5 x 10 12 to 3 x 10 12 GC/g brain, 0.5 x 10 12 to 2.5 x 10 12 GC/g brain, 1 x 10 12 to 2 x 10 12 GC/g brain, and all integers including and in between 0.1 x 10 12 GC/g brain to 3 x 10 12 GC/g brain) of the subject.
  • 0.1 x 10 12 to 0.5 x 10 12 GC/g brain e.g., 0.1 x 10 12 to 0.5 x 10 12 GC/g brain
  • the pharmaceutical formulation is dosed at 0.1 x 10 12 GC/g brain to 3 x 10 12 GC/g brain. In some embodiments, the pharmaceutical formulation is dosed at 0.1 x 10 12 to 0.5 x 10 12 GC/g brain. In some embodiments, the pharmaceutical formulation is dosed at 0.5 x 10 12 to 1 x 10 12 GC/g brain. In some embodiments, the pharmaceutical formulation is dosed at 1 x 10 12 to 1.5 x 10 12 GC/g brain. In some embodiments, the pharmaceutical formulation is dosed at 1.5 x 10 12 to 2 x 10 12 GC/g brain. In some embodiments, the pharmaceutical formulation is dosed at 2 x 10 12 to 2.5 x 10 12 GC/g brain.
  • the pharmaceutical formulation is dosed at 2.5 x 10 12 to 3 x 10 12 GC/g brain. In some embodiments, the pharmaceutical formulation is dosed at 0.5 x 10 12 to 2.5 x 10 12 GC/g brain. In some embodiments, the pharmaceutical formulation is dosed at 1 x 10 12 to 2 x 10 12 GC/g brain. In some embodiments, the pharmaceutical formulation is dosed at 1 x 10 12 GC/g brain of the subject.
  • the pharmaceutical formulation comprises sodium phosphate, sodium chloride (NaCl), sorbitol, poloxamer 188, potassium chloride (KC1), magnesium chloride (MgCh), calcium chloride (CaC12), or a combination thereof.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 1% to at least 20% (e.g., at least 1% to at least 1.5% , at least 1.5% to at least 2%, at least 2% to at least 2.5%, at least 2.5% to at least 3%, at least 3% to at least 3.5%, at least 3.5% to at least 4%, at least 4% to at least 4.5%, at least 4.5% to at least 5%, at least 5% to at least 5.5%, at least 5.5% to at least 6%, at least 6% to at least 6.5%, at least 6.5% to at least 7%, at least 7% to at least 7.5%, at least 7.5% to at least 8%, at least 8% to at least 8.5%, at least 8.5% to at least 9%, at least 9% to at least 9.5%, at least 9.5% to at least 10%, at least 10% to at least 10.5%, at least 10.5% to at least 11 %, at least 11 % to at least 11.5%, at least 11.5% to at least
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 1% to at least 20% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 1% to at least 1.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 1.5% to at least 2% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 2% to at least 2.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 2.5% to at least 3% of the total formulation.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 3% to at least 3.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 3.5% to at least 4% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 4% to at least 4.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 4.5% to at least 5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 5% to at least 5.5% of the total formulation.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 5.5% to at least 6% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 6% to at least 6.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 6.5% to at least 7% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 7% to at least 7.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 7.5% to at least 8% of the total formulation.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 8% to at least 8.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 8.5% to at least 9% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 9% to at least 9.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 9.5% to at least 10% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 10% to at least 10.5% of the total formulation.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 10.5% to at least 11% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 11% to at least 11.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 11.5% to at least 12% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 12% to at least 12.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 12.5% to at least 13% of the total formulation.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 13% to at least 13.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 13.5% to at least 14% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 14% to at least 14.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 14.5% to at least 15% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 15% to at least 15.5% of the total formulation.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 15.5% to at least 16% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 16% to at least 16.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 16.5% to at least 17% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 17% to at least 17.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 17.5% to at least 18% of the total formulation.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 18% to at least 18.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 18.5% to at least 19% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 19% to at least 19.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 19.5% to at least 20% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 1.5% to at least 19.5% of the total formulation.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 2% to at least 19% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 2.5% to at least 18.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 3% to at least 18% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 3.5% to at least 17.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 4% to at least 17% of the total formulation.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 4.5% to at least 16.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 5% to at least 16% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 5.5% to at least 15.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 6% to at least 15% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 7% to at least 14.5% of the total formulation.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 7.5% to at least 14% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 8% to at least 13.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 8.5% to at least 13% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 9% to at least 12.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 9.5% to at least 12% of the total formulation.
  • the pharmaceutical formulation comprises sorbitol at a concentration of at least 10% to at least 11.5% of the total formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 5% to at least 10% of the total volume of the formulation. In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of at least 5% of the total volume of the formulation.
  • the pharmaceutical formulation comprises: (i) 10 mM sodium phosphate, 135 mMNaCl, 0.27M Sorbitol, 0.001% v/v poloxamer 188, pH 7.2; (ii) 10 mM sodium phosphate, 135 mM NaCl, 0.54M Sorbitol, 0.001% v/v poloxamer 188, pH 7.2; or (iii) 10 mM Tris, 130 mM NaCl, 2 mM KC1, 1 mM MgCh, 2 mM CaCh, 0.27M Sorbitol, 0.001% v/v poloxamer 188, pH 6.8.
  • the pharmaceutical formulation comprises 10 mM sodium phosphate, 135 mM NaCl, 0.27M Sorbitol, 0.001% v/v poloxamer 188, pH 7.2.
  • the pharmaceutical formulation is administered intrathecally, intracerebroventricularly, or via an intracisternal magna route.
  • the administration may be a bolus injection or it may be a continuous infusion.
  • the pharmaceutical formulation is provided in a vial holding 1 mL to 20 mL (e.g., 1 mL to 4 mL, 4 mL to 8 mL, 8 mL to 12 mL, 12 mL to 16 mL, 16 mL to 20 mL, 4 mL to 16 mL, 8 mL to 16 mL, 12 mL to 20 mL, and all integers including and in between 1 mL to 20 mL).
  • the pharmaceutical formulation is provided in a vial holding 1 mL to 20 mL.
  • the pharmaceutical formulation is provided in a vial holding 1 mL to 4 mL.
  • the pharmaceutical formulation is provided in a vial holding 4 mL to 8 mL. In some embodiments, the pharmaceutical formulation is provided in a vial holding 8 mL to 12 mL. In some embodiments, the pharmaceutical formulation is provided in a vial holding 12 mL to 16 mL. In some embodiments, the pharmaceutical formulation is provided in a vial holding 16 mL to 20 mL. In some embodiments, the pharmaceutical formulation is provided in a vial holding 4 mL to 16 mL. In some embodiments, the pharmaceutical formulation is provided in a vial holding 8 mL to 16 mL. In some embodiments, the pharmaceutical formulation is provided in a vial holding 12 mL to 20 mL.
  • the pharmaceutical formulation is provided in a vial holding 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL.
  • the pharmaceutical formulation is provided in a vial holding 1 mL.
  • the pharmaceutical formulation is provided in a vial holding 2 mL.
  • the pharmaceutical formulation is provided in a vial holding 3 mL.
  • the pharmaceutical formulation is provided in a vial holding 4 mL.
  • the pharmaceutical formulation is provided in a vial holding 5 mL.
  • the pharmaceutical formulation is provided in a vial holding 6 mL. In some embodiments, the pharmaceutical formulation is provided in a vial holding 7 mL. In some embodiments, the pharmaceutical formulation is provided in a vial holding 8 mL. In some embodiments, the pharmaceutical formulation is provided in a vial holding 9 mL. In some embodiments, the pharmaceutical formulation is provided in a vial holding 10 mL.
  • the pharmaceutical formulation is dosed in an amount ranging from 0.5 mL to 12 mL (e.g., 0.5 mL to 1 ml, 1 mL to
  • the pharmaceutical formulation is dosed in an amount ranging from 0.5 mL to 1 mL. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 1 mL to 1.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 1.5 mL to 2 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 2 mL to 2.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 2.5 mL to 3 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 3 mL to 3.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 3.5 mL to 4 ml.
  • the pharmaceutical formulation is dosed in an amount ranging from 4 mL to 4.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 4.5 mL to 5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 5 mL to 5.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 5.5 mL to 6 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 6 mL to 6.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 6.5 mL to 7 ml.
  • the pharmaceutical formulation is dosed in an amount ranging from 7 mL to 7.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 7.5 mL to 8 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 8 mL to 8.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 8.5 mL to 9 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 9 mL to 9.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 9.5 mL to 10 ml.
  • the pharmaceutical formulation is dosed in an amount ranging from 10 mL to 10.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 10.5 mL to 11 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 11 mL to 11.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 11.5 mL to 12 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 1 ml to 11.5 mL. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 1.5 ml to 11 ml.
  • the pharmaceutical formulation is dosed in an amount ranging from 2 ml to 10.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 2.5 ml to 10 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 3 ml to 9.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 3.5 ml to 9 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 4 ml to 8.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 4.5 ml to 8 ml.
  • the pharmaceutical formulation is dosed in an amount ranging from 5 ml to 7.5 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 5.5 ml to 7 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 6 ml to 7 ml. In some embodiments, the pharmaceutical formulation is dosed in an amount ranging from 2 mL to 8 mL.
  • the CNS disorder is selected from CDKL5 deficiency disorder (CDD), Angelman syndrome, Batten disease, Krabbe disease, Parkinson's disease, Alzheimer's disease, Spinal Muscular Atrophy (SMA) Types I, II, III, or IV, X-linked Myotubular Myopathy, Friedrich's Ataxia, Canavan's disease, Amyotrophic Lateral Sclerosis (ALS), Adrenoleukodystrophy, Huntington disease, Rett syndrome, or Spinocerebellar ataxia.
  • CDD CDKL5 deficiency disorder
  • Angelman syndrome Batten disease
  • Krabbe disease Krabbe disease
  • Parkinson's disease Alzheimer's disease
  • SMA Spinal Muscular Atrophy
  • Types I, II, III, or IV Types I, II, III, or IV
  • X-linked Myotubular Myopathy Friedrich's Ataxia
  • Canavan's disease Amyotrophic Lateral Sclerosis (ALS), Adrenoleukodystrophy
  • Huntington disease
  • the rAAV comprises an AAV capsid and a vector genome packaged therein.
  • the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rhlO, hu37 (z.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAVrhlO, AAVhu37), or an engineered variant thereof.
  • the AAV capsid is an AAV serotype 9 (AAV9) capsid, an AAV9 variant capsid, an AAV serotype 8 (AAV8) capsid, an AAV8 variant capsid, or an AAV serotype hu37 (AAVhu37) capsid.
  • the CNS disorder is CDD and the rAAV comprises a nucleic acid sequence encoding a CDKL5 polypeptide.
  • the CNS disorder is CDD and the rAAV is described in WO/2021/163322.
  • the CNS disorder is CDD and the rAAV comprises an AAV9 capsid and a vector genome comprising SEQ ID NO: 1.
  • the present disclosure provides a process for preparing a pharmaceutical formulation for administration to the CNS of a subject suffering from a CNS disorder, wherein the pharmaceutical formulation comprises (i) a drug substance present at a minimum drug substance concentration, (ii) a maximum dose volume, and (iii) a maximum endotoxin concentration.
  • the process comprises: a) selecting a delivery route for the pharmaceutical formulation from intrathecal (IT), intracerebroventricular (ICV), and intra cisterna magna (ICM); b) based on the selected delivery route, selecting one or more excipients for the pharmaceutical formulation selected from a salt and/or a buffer; c) determining whether one or more of the excipients has a deleterious effect on one or more physical property of the pharmaceutical formulation selected from stability of the drug substance, viscosity, salt concentration, conductivity, trace metal content, osmolarity, density, pH, and biodistribution of the drug substance in the brain of a subject administered the pharmaceutical formulation; d) increasing the concentration of the one or more excipients in the pharmaceutical formulation to facilitate increasing drug substance concentration of the formulation if needed prior to purifying and/or concentrating the drug substance or spiking one or more of the excipients into the formulation after purifying and/or concentrating the drug substance; e) selecting low endotoxin
  • the process disclosed herein results in extended stability of the pharmaceutical formulation.
  • the pharmaceutical formulation demonstrates stability for at least 2 months to at least 24 months (e.g., 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months).
  • the pharmaceutical formulation demonstrates stability for at least 12 months.
  • the pharmaceutical formulation demonstrates stability for at least 2 months to at least 24 months (e.g., 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months) in long term storage (e.g., ⁇ -60 °C).
  • long term storage e.g., ⁇ -60 °C
  • the pharmaceutical formulation demonstrates stability for at least 12 months in long term storage at ⁇ -60 °C.
  • the drug substance is a recombinant adeno-associated vector (rAAV).
  • the maximum dose volume is 12 mL.
  • the maximum endotoxin concentration is 0.2 EU/mL.
  • the present disclosure provides a process for preparing a pharmaceutical formulation (comprising, e.g., a recombinant adeno-associated vector (rAAV)) for administration to the CNS of a subject.
  • a pharmaceutical formulation comprising, e.g., a recombinant adeno-associated vector (rAAV)
  • rAAV recombinant adeno-associated vector
  • the pharmaceutical formulation may comprise (i) a drug substance present at a minimum drug substance concentration, (ii) a maximum dose volume, and (iii) a maximum endotoxin concentration.
  • the process for preparing the pharmaceutical formulation comprises selecting a delivery route for the pharmaceutical formulation from intrathecal (IT), intracerebroventricular (ICV), and intra cisterna magna (I CM), then, based on the selected delivery route, selecting one or more excipients for the pharmaceutical formulation from a salt and/or a buffer. Having selected delivery route and identified appropriate excipients, the process may further entail determining whether one or more of the excipients has a deleterious effect on one or more physical property of the pharmaceutical formulation.
  • Physical properties to be considered include stability of the drug substance, viscosity, salt concentration, conductivity, trace metal content, osmolarity, density, pH, and biodistribution of the drug substance in the brain of a subject administered the pharmaceutical formulation.
  • the excipient(s) should be selected to have minimal deleterious effect(s).
  • the process further comprises selecting low endotoxin raw materials for use in drug substance and pharmaceutical formulation manufacturing processes. In some embodiments, the process further comprises evaluating strategies for further lowering raw materials endotoxins by cleaning and depyrogenation steps, wherein key raw materials endotoxin levels are ⁇ 0.1 EU/mL for buffers, ⁇ 0.01 EU/mL for pharmaceutical formulation vials, and/or ⁇ 0.05 EU/mL for each piece of tubing or container that will contact the drug substance or the pharmaceutical formulation. In some embodiments of the processes of the present disclosure, low endotoxin raw materials are selected for use in dose preparation.
  • the process of the present disclosure further comprises selecting raw materials and components for use in dose preparation and administration, such that endotoxin concentration of the formulation is calculated according to Formula I:
  • eop is the endotoxin concentration of the drug product
  • EU is endotoxin units
  • mbody is body weight of the subject
  • Eoev is total endotoxin contribution from components used for dose preparation and dose administration of the formulation
  • mbrain is brain weight of the subject
  • Dbrain is the dose to be delivered to CNS based on the mbrain and mbody of subject, e.g., as disclosed in Table 4
  • CDP is drug product concentration
  • 0.2 EU/kg is the tolerable endotoxin limit per body weight for Intrathecal Bolus Dose ( ⁇ Bit) (as per USP ⁇ 85>).
  • eop may be equivalent to EDP (total endotoxin contribution of the drug product determined by adding the endotoxin contribution from the raw materials comprising the one or more excipients and the drug product) divided by VDP (volume of the drug product), .
  • low endotoxin raw materials are alternatively or additionally implemented in dose administration to the subject to maximally reduce endotoxin exposure to the subject administered the pharmaceutical formulation.
  • the drug substance is a rAAV
  • the minimum CDP is 1 X 10 14 to 3 x 10 14 GC/ml (e.g., 1 x 10 14 to 1.2 x 10 14 GC/ml, 1.2 x 10 14 to 1.4 x 10 14 GC/ml, 1.4 x 10 14 to 1.6 x 10 14 GC/ml, 1.6 x 10 14 to 1.8 x 10 14 GC/ml, 1.8 x 10 14 to 2 x 10 14 GC/ml, 2 x 10 14 to 2.2 x 10 14 GC/ml, 2.2 x 10 14 to 2.4 x 10 14 GC/ml, 2.4 x 10 14 to 2.6 x 10 14 GC/
  • the drug substance is a rAAV, and the minimum CDP is 1 X 10 14 to 1.2 x 10 14 GC/ml. In some embodiments of the process of the present disclosure, the drug substance is a rAAV, and the minimum CDP is 1.2 x 10 14 to 1.4 x 10 14 GC/ml. In some embodiments of the process of the present disclosure, the drug substance is a rAAV, and the minimum CDP is 1.4 x 10 14 to 1.6 x 10 14 GC/ml. In some embodiments of the process of the present disclosure, the drug substance is a rAAV, and the minimum CDP is 1.6 x 10 14 to 1.8 x 10 14 GC/ml.
  • the drug substance is a rAAV, and the minimum CDP is 1.8 x 10 14 to 2 x 10 14 GC/ml. In some embodiments of the process of the present disclosure, the drug substance is a rAAV, and the minimum CDP is 2 X 10 14 to 2.2 x 10 14 GC/ml. In some embodiments of the process of the present disclosure, the drug substance is a rAAV, and the minimum CDP is 2.2 x 10 14 to 2.4 x 10 14 GC/ml. In some embodiments of the process of the present disclosure, the drug substance is a rAAV, and the minimum CDP is 2.4 x 10 14 to 2.6 x 10 14 GC/ml.
  • the drug substance is a rAAV, and the minimum CDP is 2.6 X 10 14 to 2.8 x 10 14 GC/ml. In some embodiments of the process of the present disclosure, the drug substance is a rAAV, and the minimum CDP is 2.8 x 10 14 to 3 x 10 14 GC/ml. In some embodiments of the process of the present disclosure, the drug substance is a rAAV, and the minimum CDP is 1.2 x 10 14 to 3 x 10 14 GC/ml. In some embodiments of the process of the present disclosure, the drug substance is a rAAV, and the minimum CDP is 1.4 x 10 14 to 2.8 x 10 14 GC/ml.
  • the drug substance is a rAAV, and the minimum CDP is 1.6 x 10 14 to 2.6 x 10 14 GC/ml. In some embodiments of the process of the present disclosure, the drug substance is a rAAV, and the minimum CDP is 1.8 x 10 14 to 2.4 x 10 14 GC/ml. In some embodiments of the process of the present disclosure, the drug substance is a rAAV, and the minimum CDP is at least 2 x 10 14 GC/ml in the pharmaceutical formulation.
  • the formulation comprises a total rAAV dose of 3.6 x 10 14 to 3 x 10 15 GC (e.g., 3.6 x 10 14 to 4 x 10 14 GC, 4 x 10 14 to 5 x 10 14 GC, 5 x 10 14 to 6 x 10 14 GC, 6 x 10 14 to 7 x 10 14 GC, 7 x 10 14 to 8 x 10 14 GC, 8 x 10 14 to 9 x 10 14 GC, 9 x 10 14 to 1 x 10 15 GC, 1 x 10 15 to 2 x 10 15 GC, 2 x 10 15 to 3 x 10 15 GC, 4 x 10 15 to 2 x 10 15 GC, 5 x 10 14 to 1 x 10 15 GC, 6 x 10 14 to 9 x 10 14 GC, and all integers including and in between 3.6 x 10 14 to 3 x 10 15 GC).
  • 3.6 x 10 14 to 4 x 10 14 GC 4 x 10 14 to 5 x 10 14 GC,
  • the formulation comprises a total rAAV dose of 3.6 x 10 14 to 4 x 10 14 GC. In some embodiments of the process of the present disclosure, the formulation comprises a total rAAV dose of 4 x 10 14 to 5 x 10 14 GC. In some embodiments of the process of the present disclosure, the formulation comprises a total rAAV dose of 5 x 10 14 to 6 x 10 14 GC. In some embodiments of the process of the present disclosure, the formulation comprises a total rAAV dose of 6 x 10 14 to 7 x 10 14 GC. In some embodiments of the process of the present disclosure, the formulation comprises a total rAAV dose of 7 x 10 14 to 8 x 10 14 GC.
  • the formulation comprises a total rAAV dose of 8 x 10 14 to 9 x 10 14 GC. In some embodiments of the process of the present disclosure, the formulation comprises a total rAAV dose of 9 x 10 14 to 1 x 10 15 GC. In some embodiments of the process of the present disclosure, the formulation comprises a total rAAV dose of 1 x 10 15 to 2 x 10 15 GC. In some embodiments of the process of the present disclosure, the formulation comprises a total rAAV dose of 2 x 10 15 to 3 x 10 15 GC. In some embodiments of the process of the present disclosure, the formulation comprises a total rAAV dose of 4 x 10 15 to 2 x 10 15 GC.
  • the formulation comprises a total rAAV dose of 5 x 10 14 to 1 x 10 15 GC. In some embodiments of the process of the present disclosure, the formulation comprises a total rAAV dose of 6 x 10 14 to 9 x 10 14 GC.
  • the pharmaceutical formulation is dosed at 0.1 x 10 12 GC/g brain to 3 x 10 12 GC/g brain (e.g., 0.1 x 10 12 to 0.5 x 10 12 GC/g brain, 0.5 x 10 12 to 1 x 10 12 GC/g brain, 1 x 10 12 to 1.5 x 10 12 GC/g brain, 1.5 x 10 12 to 2 x 10 12 GC/g brain, 2 x 10 12 to 2.5 x 10 12 GC/g brain, 2.5 x 10 12 to 3 x 10 12 GC/g brain, 0.5 x 10 12 to 2.5 x 10 12 GC/g brain, 1 x 10 12 to 2 x 10 12 GC/g brain, and all integers including and in between 0.1 x 10 12 to 3 x 10 12 GC/g brain) of the subject.
  • 0.1 x 10 12 to 0.5 x 10 12 GC/g brain e.g., 0.1 x 10 12 to 0.5 x 10 12 GC/g brain, 0.5 x
  • the pharmaceutical formulation is dosed at 0.1 x 10 12 to 0.5 x 10 12 GC/g brain of the subject. In some embodiments of the process of the present disclosure, the pharmaceutical formulation is dosed at 0.5 x 10 12 to 1 x 10 12 GC/g brain of the subject. In some embodiments of the process of the present disclosure, the pharmaceutical formulation is dosed at 1 x 10 12 to 1.5 x 10 12 GC/g brain of the subject. In some embodiments of the process of the present disclosure, the pharmaceutical formulation is dosed at 1.5 x 10 12 to 2 x 10 12 GC/g brain of the subject.
  • the pharmaceutical formulation is dosed at 2 x 10 12 to 2.5 x 10 12 GC/g brain of the subject. In some embodiments of the process of the present disclosure, the pharmaceutical formulation is dosed at 2.5 x 10 12 to 3 x 10 12 GC/g brain of the subject. In some embodiments of the process of the present disclosure, the pharmaceutical formulation is dosed at 0.5 x 10 12 to 2.5 x 10 12 GC/g brain of the subject. In some embodiments of the process of the present disclosure, the pharmaceutical formulation is dosed at 1 x 10 12 to 2 x 10 12 GC/g brain of the subject. In some embodiments, the pharmaceutical formulation is dosed at 1 x 10 12 GC/g brain of the subject.
  • the mbrain is 300g- 1400g.
  • the mbrain is 500g-700g (e.g., 500g-550g, 550g-600g, 600g- 650g, 650g-700g, 550g-650g, 600g-700g, and all integers including and in between 500g-700g).
  • the mbrain is 500g-550g.
  • the mbrain is 550g-600g.
  • the mbrain is 600g-650g.
  • the mbrain is 650g-700g.
  • the mbrain is 550g-650g. In some embodiments of this process, the mbrain is 600g-700g In some embodiments of the process of the present disclosure, the mbody is 2 kg to 25 kg (e.g., 2 kg to 5 kg, 5 kg to 7 kg, 7 kg to 9 kg, 9 kg to 12 kg, 12 kg to 15 kg, 15 kg to 20 kg, 20 kg to 25 kg, 5 kg to 20 kg, 7 kg to 15 kg, 9 kg to 15 kg, 12 kg to 20 kg, and all integers including and in between 2 kg to
  • the mbody is 2 kg to 5 kg. In some embodiments of the process of the present disclosure, the mbody is 5 kg to 7 kg. In some embodiments of the process of the present disclosure, the mbody is 7 kg to 9 kg. In some embodiments of the process of the present disclosure, the mbody is 9 kg to 12 kg. In some embodiments of the process of the present disclosure, the mbody is 12 kg to 15 kg. In some embodiments of the process of the present disclosure, the mbody is 15 kg to 20 kg. In some embodiments of the process of the present disclosure, the mbody is 20 kg to 25 kg. In some embodiments of the process of the present disclosure, the mbody is 5 kg to 20 kg.
  • the mbody is 7 kg to 15 kg. In some embodiments of the process of the present disclosure, the mbody is 9 kg to 15 k. In some embodiments of the process of the present disclosure, the mbody is 12 kg to 20 kg. In some embodiments of the process of the present disclosure, the mbody is 3 kg to 6 kg (e.g., 3 kg to 3.5 kg, 3.5 kg to 4 kg, 4 kg to 4.5 kg, 4.5 kg to 5 kg, 5.5 kg to 6 kg, 3.5 kg to 5.5 kg, 4 kg to 5 kg, 3.5 kg to 5.5 kg, 4.5 kg to 5.5 kg, and all integers including and in between 3 kg to 6 kg).
  • 3 kg to 6 kg e.g., 3 kg to 3.5 kg, 3.5 kg to 4 kg, 4 kg to 4.5 kg, 4.5 kg to 5 kg, 5.5 kg to 6 kg, 3.5 kg to 5.5 kg, 4 kg to 5 kg, 3.5 kg to 5.5 kg, 4.5 kg to 5.5 kg, and all integers including and in between 3 kg to
  • the mbody is 3 kg to 3.5 kg. In some embodiments of the process of the present disclosure, the mbody is 3.5 kg to 4 kg. In some embodiments of the process of the present disclosure, the mbody is 4 kg to 4.5 kg. In some embodiments of the process of the present disclosure, the mbody is 4.5 kg to 5 kg. In some embodiments of the process of the present disclosure, the mbody is 5.5 kg to 6 kg. In some embodiments of the process of the present disclosure, the mbody is 3.5 kg to 5.5 kg. In some embodiments of the process of the present disclosure, the mbody is 4 kg to 5 kg. In some embodiments of the process of the present disclosure, the mbody is 3.5 kg to 5.5 kg. In some embodiments of the process of the present disclosure, the mbody is 4.5 kg to 5.5 kg.
  • the VDP is 1.8 to 12 ml (e.g., 1.8 to 2.4 ml, 2.4 to 4.8 ml, 4.8 to 7.5 ml, 7.5 to 10 ml, 10 to 12 ml, 2.4 to 10 ml, 4.8 ml to 10 ml, 2.4 ml to 7.5 ml, 7.5 ml to 12 ml, and all values to the one-tenth decimal value including and in between 1.8 ml to 12 ml).
  • the VDP is 1.8 to 2.4 ml.
  • the VDP is 2.4 to 4.8 ml. In some embodiments of the process of the present disclosure, the VDP is 4.8 to 7.5 ml. In some embodiments of the process of the present disclosure, the VDP is 7.5 to 10 ml. In some embodiments of the process of the present disclosure, the VDP is 10 to 12 ml. In some embodiments of the process of the present disclosure, the VDP is 2.4 to 10 ml. In some embodiments of the process of the present disclosure, the VDP is 4.8 ml to 10 ml. In some embodiments of the process of the present disclosure, the VDP is 2.4 ml to 7.5 ml. In some embodiments of the process of the present disclosure, the VDP is 7.5 ml to 12 ml. In some embodiments of the process of the present disclosure, the VDP is 10 ml.
  • the maximum endotoxin concentration of the pharmaceutical formulation is ⁇ 0.2 EU/ml. In some embodiments of the process of the present disclosure, the maximum endotoxin concentration of the pharmaceutical formulation is ⁇ 0.1 EU/ml.
  • the eop endotoxin limit is 0.08 to 0.74 EU/ml (e.g., 0.08 to 0.15 EU/ml, 0.15 to 0.3 EU/ml, 0.3 to 0.5 EU/ml, 0.5 to 0.74 EU/ml, 0.15 to 0.5 EU/ml, 0.3 to 0.74 EU/ml, 0.15 to 0.5 EU/ml, and all values to the one- hundredth decimal value including and in between 0.08 to 0.74 EU/ml). In some embodiments of the process of the present disclosure, the eop endotoxin limit is 0.08 to 0.15 EU/ml.
  • the eop endotoxin limit is 0.15 to 0.3 EU/ml. In some embodiments of the process of the present disclosure, the eop endotoxin limit is 0.3 to 0.5 EU/ml. In some embodiments of the process of the present disclosure, the eop endotoxin limit is 0.5 to 0.74 EU/ml. In some embodiments of the process of the present disclosure, the eop endotoxin limit is 0.15 to 0.5 EU/ml. In some embodiments of the process of the present disclosure, the eop endotoxin limit is 0.3 to 0.74 EU/ml. In some embodiments of the process of the present disclosure, the eop endotoxin limit is 0.15 to 0.5 EU/ml.
  • the Edev is up to 0.32 EU. In some embodiments, the process of the present disclosure further comprises lowering the Edev by cleaning and depyrogenating the components used for dose preparation and dose administration of the formulation.
  • the Edev comprises ⁇ 0.01 EU/mL for pharmaceutical formulation vials, and/or ⁇ 0.05 EU/mL for each piece of tubing or container that will contact the drug substance or the pharmaceutical formulation.
  • the Edev comprises ⁇ 0.1 EU/mL for at least one buffer used as raw material.
  • the rAAV comprises an AAV capsid and a vector genome packaged therein.
  • the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rhlO, hu37 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAVrhlO, AAVhu37), or an engineered variant thereof.
  • the CNS disorder is CDD and the rAAV comprises a nucleic acid sequence encoding a CDKL5 polypeptide.
  • the CNS disorder is CDD and the rAAV is described in WO/2021/163322.
  • the CNS disorder is CDD and the rAAV comprises an AAV9 capsid and a vector genome comprising SEQ ID NO: 1.
  • FIG. 1 is a graph showing diffusion coefficient analysis of rAAV concentration in presence of salt only (100 mM NaCl) or salt with additive (130 mM NaCl and 5% sorbitol). The diffusion coefficient is indicated on the y-axis and the concentration of rAAV in terms of genomic titer is indicated on the x-axis.
  • FIGs. 2A-2D are histogram plots evaluating genome titer at 37 °C (FIG. 2A), in vitro mRNA expression (FIG. 2B; F2 was not tested for in vitro mRNA expression for the 1 Ox freezethaw cycle, as indicated), capsid impurity level when subjected to capillary electrophoresis (CE)- SDS (FIG. 2C), and percentage of high molecular weight aggregation determined using sizeexclusion chromatography (SEC) (FIG.
  • CE capillary electrophoresis
  • SEC sizeexclusion chromatography
  • the three tested formulations were Fl (10 mM sodium phosphate, 135 mM NaCl, 0.27M Sorbitol, 0.001% v/v poloxamer 188, pH 7.2); F2 (10 mM sodium phosphate, 135 mM NaCl, 0.54M Sorbitol, 0.001% v/v poloxamer 188, pH 7.2); and F3 (10 mM Tris, 130 mM NaCl, 2 mM KC1, 1 mM MgC12, 2 mM CaC12, 0.27M Sorbitol, 0.001% v/v poloxamer 188, pH 6.8).
  • FIGs. 3A-3D are fluorescence microscopy images showing effect on CDKL5 mRNA expression in brain tissue of animals injected with the rAAV-CDKL5 in the three different formulations disclosed herein as follows: Fl: 10 mM sodium phosphate, 135 mM NaCl, 0.27M Sorbitol, 0.001% v/v poloxamer 188, pH 7.2 (FIG. 3B); F2: 10 mM sodium phosphate, 135 mM NaCl, 0.54M Sorbitol, 0.001% v/v poloxamer 188, pH 7.2 (FIG.
  • F3 10 mM Tris, 130 mM NaCl, 2 mM KC1, 1 mM MgCh, 2 mM CaCh, 0.27M Sorbitol, 0.001% v/v poloxamer 188, pH 6.8 (FIG. 3 A), and Fl with hydroxychloroquine at 25 mg/kg (FIG. 3D).
  • the expression of CDKL5 in brain tissue is indicated by arrows. .
  • FIG. 4 is a chart showing the outline of drug substance manufacturing and drug product manufacturing enabling tight control of AAV concentration.
  • Abbreviation used in FIG. 4 formulation tangential flow filtration (FTFF); tangential flow filtration (TFF); at status quantitative PCR (STAT qPCR); drug substance (DS); drug product (DP); droplet digital PCR (ddPCR).
  • FIG. 5 is a chart showing estimated dose volume of drug product per minimum patient’s body weight (ml/kg) as indicated on y-axis corresponding to patient age as indicated on x-axis. The low and high brain weight groups are as indicated.
  • FIG. 6 is a graph showing the contribution of drug product (DP) endotoxin level to the endotoxin risk of patients administered drug dosage based on body weight.
  • the assumed drug product concentration is IxlO 14 GC/ml
  • the device endotoxin contribution is 0.5 endotoxin unit (EU)/device.
  • the total endotoxin deliverable to patients corresponding to body weight, based on the USP ⁇ 85> EU limit of 0.2 EU/Kg is indicated as solid black line.
  • FIG. 7 is a graph showing the contribution of endotoxin from device to the endotoxin risk of patients administered drug dosage based on body weight.
  • the assumed drug product concentration is 1x10 14 GC/ml, and the drug product endotoxin contribution is 0.1 EU/ml.
  • the total endotoxin deliverable to patients corresponding to body weight, based on the EU limit of 0.2 EU/Kg is indicated as solid black line.
  • FIG. 8 is a graph showing the contribution of endotoxin from device to the endotoxin risk of patients administered drug dosage based on body weight.
  • the assumed high dose of the drug product is 1.2xl0 12 GC/g brain weight, drug product concentration of IxlO 14 GC/ml, measured endotoxin concentration of the drug product (eDP) is ⁇ 0.1 EU/ml and the endotoxin contribution from the device is ⁇ 0.2601 EU/device.
  • the total endotoxin deliverable to patients corresponding to body weight, based on the USP ⁇ 85> EU limit of 0.2 EU/Kg is indicated as solid black line.
  • EU total endotoxin
  • CDKL5 deficiency disorder a rare neurodevelopmental disease caused by mutations in the CDKL5 gene which can manifest in a broad range of clinical symptoms and severity.
  • Hallmarks of CDD include infantile-onset refractory epilepsy, developmental delay, intellectual disability, visual impairment, lack of speech, hypotonia, motor dysfunction, sleep disturbances, gastrointestinal dysfunction, and breathing difficulties. Although rare, the occurrence is believed to be approximately 1 in 40,000 - 60,000 live births, making it one of the most common forms of genetic epilepsy.
  • AAV for the treatment of CDD and formulations comprising the same are described in WO/2021/163322 and WO/2023/023590, respectively, the disclosures of which are herein incorporated by reference in their entireties.
  • the applicant has determined it is important to deliver the rAAV directly to the brain, e.g., through intracisternal magna (ICM) administration, to achieve widespread distribution and neuron transduction.
  • ICM intracisternal magna
  • the applicant has also determined it is important to use a high dose of rAAV, e.g., at least about 1 x 10 12 genome copies (GC)/gram of brain tissue, which can be dose dependent based upon patient brain mass.
  • GC genome copies
  • CSF cerebrospinal fluid
  • a low dose volume ⁇ 12 mL is desirable to prevent disruption of the pressure balance in the central nervous system (CNS) region.
  • Applicant has designed formulations comprising high concentrations of recombinant AAV and low levels of endotoxin. These formulations can be successfully administered (e.g., intrathecally) to the subjects for the treatment of rare genetic disorders of the CNS.
  • the present disclosure provides methods for preparing pharmaceutical formulations comprising rAAV suitable for administration to the CNS via intrathecal (IT), intracerebroventricular (ICV), and/or intracisternal magna (ICM) routes.
  • pharmaceutical formulations comprising rAAV suitable for administration to the CNS and methods of their use in the treatment of CNS disorders, e.g., rare genetic CNS disorders such as CDKL5 deficiency disorder (CDD).
  • CDKL5 deficiency disorder CDKL5 deficiency disorder
  • Adeno-associated virus A small, replication-defective, non-enveloped virus that infects humans and some other primate species. AAV is not known to cause disease and elicits a very mild immune response. Gene therapy vectors that utilize AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV an attractive viral vector for gene therapy. There are currently 13 recognized serotypes of AAV (AAV1 - 13). The term “AAV particle” refers to an AAV capsid and a genome packaged therein.
  • Administration/Administer To provide or give a subject an agent, such as a therapeutic agent (e.g., a recombinant AAV), by any effective route.
  • routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal (ID), intraperitoneal (IP), intrathecal (IT), intracerebroventricular (ICV), intracisternal magna (ICM), or intravenous (IV) administration), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • Central nervous system generally refers to the spinal cord and brain and contrasts with the "peripheral nervous system" which excludes the spinal cord and brain.
  • the central nervous system There are different cell types within the central nervous system, including neuronal cells and glial cells.
  • the glia in mature systems include astrocytes, oligodendrocytes, and microglial cells.
  • the AAV capsids for the vectors used in the present invention are preferably selected from among those which will transduce and/or express in at least one of these cell types of the central nervous system.
  • Endotoxin generally refers to toxic substance(s) composed of lipopolysaccharides (LPS), which comprises a lipid portion (lipid A), a core polysaccharide, and an O-specific polysaccharide chain.
  • LPS lipopolysaccharides
  • Endotoxins are released when a bacterial cell wall is disrupted, such as during bacterial lysis, growth, or death. Because endotoxins may stimulate the innate immune system, their removal from medical products, especially injectable drugs and medical devices, is important.
  • Methods for determining the amount of endotoxin are known in the art, e.g., one may use a limulus amoebocyte lysate (LAL) test. For instance, the amount of endotoxin is measured using a traditional LAL kinetic chromogenic assay, e.g., such as the highly-sensitive Endochrome- KTM assay available from Charles River Laboratories International, Wilmington, MA.
  • Intrathecal delivery/Intrathecal administration generally refers to a route of administration for drugs via an injection, e.g., an infusion, into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF).
  • Intrathecal delivery may include lumbar puncture, intraventricular (including intracerebroventricular (ICV)), suboccipital/intracisternal, and/or CI-2 puncture.
  • material e.g., a pharmaceutical formulation
  • injection may be into the cisterna magna.
  • Intracisternal magna delivery/Intracisternal magna administration/inj ection into the cisterna magna generally refers to a route of administration for drugs directly into the cerebrospinal fluid of the cisterna magna cerebellomedularis, more specifically via a suboccipital puncture or by direct injection into the cisterna magna or via permanently positioned tube.
  • Preventing, treating or ameliorating a disease “Preventing” a disease (such as CDD) refers to inhibiting the full development of a disease.
  • Treating refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition (such as CDD) after it has begun to develop. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease (such as CDD).
  • a recombinant nucleic acid molecule is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques.
  • a recombinant virus is a virus comprising sequence (such as genomic sequence) that is non-naturally occurring or made by artificial combination of at least two sequences of different origin.
  • the term “recombinant” also includes nucleic acids, proteins and viruses that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule, protein or virus.
  • “recombinant AAV” also rAAV, used interchangeably with rAAV particle) refers to an AAV particle in which a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule encoding CDKL5) has been packaged.
  • Serotype A group of closely related microorganisms (such as viruses) distinguished by a characteristic set of antigens.
  • Subject Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.
  • the subject is a human.
  • the human subject is an adult subject, i.e., a human subject greater than 18 years old.
  • the human subject is a pediatric subject, i.e., a human subject of ages 0-18 years old inclusive.
  • Therapeutically effective amount A quantity of a specified pharmaceutical or therapeutic agent (e.g., a recombinant AAV) sufficient to achieve a desired effect in a subject, or in a cell, being treated with the agent.
  • the effective amount of the agent will be dependent on several factors, including, but not limited to the subject or cells being treated, and the manner of administration of the therapeutic composition.
  • Excipient Ingredients other than the active pharmaceutical ingredient (API) or drug substance present in a finished pharmaceutical drug formulation.
  • the excipient can be one or more of lubricant, diluent, binder, flavoring, buffer, coating and coloring agent for the finished pharmaceutical drug formulation.
  • the one or more excipients of the formulation are selected based on the route of administration of the finished pharmaceutical drug formulation to the subject, the concentration of the drug product in the finished pharmaceutical drug formulation, the physical properties of the drug product (e.g. capsid serotype of a rAAV drug product) within the finished pharmaceutical drug formulation, or a combination thereof.
  • endotoxin contribution from an excipient in the pharmaceutical drug formulation is the total endotoxin contribution from the raw materials/components used for making or preparing the one or more excipients, i.e., the level of total endotoxin present in the amount of the various components used to make the excipient.
  • the endotoxin contribution from a buffer (an excipient) comprising 10 mM sodium phosphate, 135 mMNaCl, 0.27M Sorbitol, 0.001% v/v poloxamer 188, is the total endotoxin amount calculated by adding up the endotoxin levels in the respective amounts of each buffer component i.e., sodium phosphate, NaCl, Sorbitol, poloxamer 188, that were combined to form the buffer.
  • each buffer component i.e., sodium phosphate, NaCl, Sorbitol, poloxamer 188
  • Drug product (DP) is a finished pharmaceutical drug formulation comprising a drug substance or API and one or more excipients.
  • a drug product may be liquid or solid, lyophilized or reconstituted in liquid, e.g., for administration.
  • Drug product or DP is used interchangeably with finished pharmaceutical drug formulation.
  • the endotoxin contribution of the drug product is the total endotoxin calculated by adding the total endotoxin from the formulation comprising the drug substance or API, in some cases a rAAV drug substance, and the one or more excipients.
  • Device components refer to machines or apparatuses and parts thereof, for preparing and administering (e.g., injecting) a drug substance within the drug product.
  • one or more endotoxins can be introduced into the drug product through one or more device components that physically contacts the one or more excipients, the drug substance, or the drug product, during preparation or administration of the drug product.
  • the endotoxin contribution from device components is calculated by adding up the endotoxin levels of each machine or apparatus or part thereof that physically contacts the one or more excipients, the drug substance, or the drug product.
  • Brain mass weight of the brain of a subject measured by methods used in the art including but not limited to magnetic resonance imaging (MRI) and computed tomography (CT) scans.
  • MRI magnetic resonance imaging
  • CT computed tomography
  • Body mass weight of the physical body of the subject measured by standing on a suitable digital or mechanical weighing scale. In some embodiments, the physical body of the subject is weighed without clothing.
  • CDP Drug product concentration
  • Dose to be delivered to CNS is the total number of genome copies of rAAV calculated by multiplying the CDP by the volume of the drug product particles that is delivered or injected into the brain of a subject.
  • a vector is a nucleic acid molecule allowing insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector can also include one or more selectable marker genes and other genetic elements.
  • An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes. In some embodiments herein, the vector is an AAV vector.
  • the term “vector” may also be used in the general sense of an agent that carries genetic information or material, for example, to introduce the genetic material to a cell or organism.
  • the term “vector” may refer to, e.g., a rAAV particle (as in the case of a “viral vector”), or to a nucleic acid molecule encoding one or more proteins of a rAAV particle (such as a plasmid), or to a nucleic acid molecule encoding a gene to be carried by a “viral vector” (i.e., a “vector genome”).
  • a rAAV particle as in the case of a “viral vector”
  • a nucleic acid molecule encoding one or more proteins of a rAAV particle such as a plasmid
  • a gene to be carried by a “viral vector” i.e., a “vector genome”.
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.
  • the range includes the specified endpoints.
  • the range “between IxlO 14 to 3xlO 15 GC/ml” includes 1x10 14 , 3x10 15 , and values greater than 1x10 14 but less than 3x10 15 .
  • the purpose of this example is to demonstrate how an rAAV-comprising pharmaceutical formulation compatible for an intracisternal magna (ICM) delivery was developed.
  • ICM intracisternal magna
  • Recombinant AAV are normally formulated with excipients such as salt and buffer.
  • excipients such as salt and buffer.
  • excipients used in clinical trials leveraging intrathecal (IT), intracerebroventricular (ICV), and intracisternal magna (ICM) delivery were identified and examined.
  • ICM intracisternal magna
  • a diffusion coefficient analysis showed that selected excipients severely limited the FTFF concentration process (FIG. 1), as evident from the non-linear behavior in sorbitol containing formulation at high rAAV concentration of 5x10 14 GC/ml and above.
  • the increase in diffusion coefficient showed a linear relationship to the increasing genome titer, in formulations without the excipient, sorbitol.
  • the excipient spike step should be performed after the concentration step.
  • Fl, F2, and F3 were evaluated for various properties indicative of stability, including genome titer, in vitro mRNA expression, integrity when subjected to capillary electrophoresis (CE)-SDS, and percent aggregation as determined by size exclusion chromatography.
  • the components of the Fl, F2, and F3 formulations were as follows:
  • Fl 10 mM sodium phosphate, 135 mM NaCl, 0.27M Sorbitol, 0.001% v/v poloxamer 188, pH 7.2.
  • F2 10 mM sodium phosphate, 135 mM NaCl, 0.54M Sorbitol, 0.001% v/v poloxamer
  • F3 10 mM Tris, 130 mMNaCl, 2 mMKCl, 1 mMMgCh, 2 mM CaCh, 0.27M Sorbitol,
  • the level of acceptable endotoxin for intrathecal delivery is ⁇ 0.2 EU/kg body weight/hr.
  • the instant inventors conducted a thorough evaluation of drug substance and drug product manufacturing process components to identify which ones can contribute to unwanted endotoxin accumulation. By virtue of this evaluation, the inventors discovered the importance of selecting low endotoxin raw materials. As a general rule, for the selection of raw materials for product development, a buffer acceptance criteria of ⁇ 0.1 EU/mL was used. For DP vials, a general acceptance criteria of ⁇ 0.01 EU/mL was established. Finally, with respect to stoppers, it was determined that, generally, an acceptable level is ⁇ 0.05 EU/mL. Assessment of materials used upstream of drug product manufacturing also should be conducted. Beyond the importance of raw materials selection, the inventors assessed cleaning and wash steps to improve endotoxin clearance; depyrogenation is an important tool to further reduce endotoxin levels as needed.
  • a pharmaceutical formulation e.g., drug product
  • LOQ low limit of quantitation
  • sensitive endotoxin method leveraged a LAL-based kinetic chromogenic method (Endochrome- KTM assay available from Charles River Laboratories International, Wilmington, MA).
  • the method is highly sensitive (detection of 0.005 EU/mL).
  • the inventors used a very low dilution factor to enable a very low LOQ of ⁇ 0.01 EU/mL. It was determined that minimal test volumes and a low dilution was critical to enabling a low LOQ.
  • Combination 1 SmartSite Universal Vial Access (Carefusion, MV0400), Texium Closed Male Luer (BD, 10012241-0500), BD Quincke Spinal Needle (BD, 405074), Millex-GP 0.2 um filter (Millipore, 5LGPM33RS), Microbore extension set 6.5 in (17 cm) (Baxter, 2N3339), Female-luer to female-luer connector (Fisher Scientific, NCI 652395), and 10-mL syringe (BD 309642).
  • Combination 2 Sterile Vial Adapter 20 (West Pharmaceuticals, 8072036), Texium CSTD (BD, 10012241-0500), BD Quincke Spinal Needle (BD, 405074), Small-bore extension set 5in (13 cm) (BBraun, 471954), and HSW Softject 10-mL (HSW, RL10).
  • Combination 3 Equashield Vial Adapter (Equashield, VA-20/2), Female LL Connector (Equashield, FC-1), Reli spinal needle (Reli, SN25G351), BBraun small bore extension tubing (BBraun, 471954), 0.2 um filter (BBraun PFE2000), Normject syringe (Normject, 4100-X00V0).
  • CSTD Closed-system transfer devices
  • Table 1 below shows the estimated brain weight range, total CSF volume range, and minimum subject body weight provided by age:
  • FIG. 5 shows the estimated drug product volume (dose volume) per minimum subject weight with age. Dosing in pediatric patients requires tight control over titer, impurities, and endotoxin levels. Table 2 below illustrates the challenge faced with the pediatric patients in terms of age (0-6 months), lowest brain weight range (300-700 g) and minimum body weight, as depicted in Table 1, corresponding to low brain weight and low CSF volume that place strict constraints on volumes of drug product that can be administered, and where a high drug product concentration is important to allow for efficacious treatment. As demonstrated in the Table 2, low concentration drug products (1x10 14 GC/ml) may require greater than 20% CSF volume of drug product to be administered in the youngest patients and patients with small CSF volume and/or brain weight.
  • Table 2 Effect of Drug Product Concentration on Dose Volume as % CSF at a target dose of 1 xlO 12 GC/g brain.
  • FIG. 6 and FIG. 7 show the effect of drug product and device contribution to the endotoxin risk, respectively.
  • FIG. 8 shows the application of the measured drug product and device components and their contribution to endotoxin, thus illustrating the challenge associated with treating patients with very low body weights.
  • EDP drug product
  • Edev administration components
  • Equation. 2 The endotoxin in the drug product, EDP, is the concentration of endotoxin in drug product multiplied by the dose volume (VDP):
  • VDP dose volume
  • Dbrain intended dose multiplied by the patient’s brain mass (mbrain) divided by the concentration of rAAV in the drug product (CrAAv):
  • Equation. 2 Equation. 2 was rearranged as:
  • Equation. 3 and Equation. 5 were combined to calculate the endotoxin concentration in the drug product (instead of total endotoxin):
  • Equation. 4 and Equation. 6 were combined to incorporate the additional dosing factors:
  • Equation. 7 the following conditions that lead to the most stringent endotoxin requirements, where eDP is minimized, were identified:
  • the rAAV dose was based on brain weight (mbrain) and the intrathecal endotoxin limit (EU) was based on minimum patient body weight (mbody), such that patients with the highest brain to body weight ratio (typically, youngest and/or smallest patients) have the highest risk of endotoxin exposure.
  • EU intrathecal endotoxin limit
  • Table 4 summarizes determination of the range of endotoxin limit required (eop or K/M D p, measured as EU/ml) shown in the right most column of the table, calculated using equation 7 described in Example 2, based on the ranges of patient mbrain, minimum mbody for different age groups, intrathecal endotoxin limit (EU) corresponding to the minimum mbody based on tolerable endotoxin limit of 0.2 EU/kg as per USP ⁇ 85>, ranges of rAAV drug concentration (GC/ml), required drug dose (total GC), and total drug volume of the rAAV dose (VDP).
  • EU intrathecal endotoxin limit
  • GC/ml ranges of rAAV drug concentration
  • total GC total rAAV dose
  • VDP total drug volume of the rAAV dose

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Abstract

La présente invention concerne des procédés de préparation de formulations pharmaceutiques comprenant un virus adéno-associé recombinant (rAAV) approprié pour une administration au système nerveux central (SNC) par l'intermédiaire de voies intrathécale (IT), intracérébroventriculaire (ICV) et/ou intracisternale magna (ICM). L'invention concerne également des formulations pharmaceutiques comprenant du rAAV approprié pour une administration au SNC et des procédés d'utilisation de celles-ci dans le traitement de troubles du SNC, par exemple, de troubles du SNC génétiques rares tels que le trouble de déficience en CDKL5 (CDD).
EP24734419.5A 2023-05-18 2024-05-17 Formulations comprenant un aav recombinant et leurs procédés d'administration Pending EP4713028A1 (fr)

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JP2021530548A (ja) * 2018-07-24 2021-11-11 ボイジャー セラピューティクス インコーポレイテッドVoyager Therapeutics, Inc. 遺伝子治療製剤を生産するための系および方法
AU2019389047A1 (en) * 2018-11-30 2021-05-20 Novartis Ag AAV viral vectors and uses thereof
EP4097239A2 (fr) * 2020-01-29 2022-12-07 Voyager Therapeutics, Inc. Procédés et systèmes de production de particules d'aav
BR112022015921A2 (pt) 2020-02-14 2022-10-04 Ultragenyx Pharmaceutical Inc Terapia gênica para tratar o transtorno de deficiência de cdkl5
JP2024531296A (ja) 2021-08-18 2024-08-29 ウルトラジェニックス ファーマシューティカル インコーポレイテッド 組換えａａｖ製剤

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