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WO2025207784A1 - Constructions de thérapie génique slc6a1 - Google Patents

Constructions de thérapie génique slc6a1

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Publication number
WO2025207784A1
WO2025207784A1 PCT/US2025/021572 US2025021572W WO2025207784A1 WO 2025207784 A1 WO2025207784 A1 WO 2025207784A1 US 2025021572 W US2025021572 W US 2025021572W WO 2025207784 A1 WO2025207784 A1 WO 2025207784A1
Authority
WO
WIPO (PCT)
Prior art keywords
expression vector
slc6a1
aav
itr
seq
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
PCT/US2025/021572
Other languages
English (en)
Inventor
Hing Cheong LEE
Alexander Rotenberg
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.)
Boston Childrens Hospital
Original Assignee
Boston Childrens Hospital
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 Boston Childrens Hospital filed Critical Boston Childrens Hospital
Publication of WO2025207784A1 publication Critical patent/WO2025207784A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • 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
    • A61K48/005—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
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • SLC6A1-related disorders are characterized by developmental delay 7,8 , autism 9 , and childhood-onset epilepsy 10 (mean seizure onset is age 3.7 years).
  • Anti- epileptic medications are ineffective in patients with SLC6A1, and a cure targeting the underlying SLC6A1 LoF such as gene replacement therapy (GRT) is lacking 11 .
  • GRT gene replacement therapy
  • patients with SLC6A1 mutations are susceptible to refractory epilepsy 12 , underscoring the significance of compensatory changes that follow increased ambient GABA that may predispose a patient to epilepsy upon SLC6A1 LoF 13 .
  • expression vectors comprising the promoter sequences as described herein, linked to a transgene.
  • the expression vector comprises a viral vector, e.g., a retrovirus, adenovirus, adeno-associated virus (AAV), or lentivirus, or a recombinant bacterial or eukaryotic plasmid.
  • the AAV is selected from the group consisting of AAV2 and AAV9.
  • the transgene is human SLC6A1.
  • the human SLC6A1 transgene is at least 95% identical to SEQ ID NO:2.
  • the expression vectors further comprise one, two, or more of: a pair of inverted terminal repeats (ITRs), a woodchuck hepatitis virus posttranscriptional response element (WPRE), and/or polyadenylation sequence.
  • ITRs inverted terminal repeats
  • WPRE woodchuck hepatitis virus posttranscriptional response element
  • polyadenylation sequence in some embodiments, the expression vector comprises a nucleic acid sequence from 5’ - 3’: ITR – hNaP - hSLC6A1 - WPRE - pA – ITR.
  • the vector comprises a nucleic acid sequence from 5’ -3’: ITR – hNaP - hSLC6A1 - pA – ITR.
  • the nucleic acid sequence is at least 90%, 95%, 97%, or 99%, or is 100% identical to SEQ ID NO:3, optionally without the WPRE sequence.
  • pharmaceutical compositions comprising the expression vectors described herein in a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions comprise a nucleic acid sequence (e.g., a vector) as described herein in a capsid, e.g., an AAV2 or AAV9 capsid.
  • the expression vector is enclosed in a AAV-BI-hTFR1 capsid, or other capsids with affinity for the human transferrin receptor (TFRC) or is enclosed in AAV-derived capsids or nanoparticles with affinity for components of the human blood-brain barrier, or otherwise have the capacity for crossing the human blood brain barrier.
  • TFRC human transferrin receptor
  • methods of treating a subject who has a SLC6A1-related disorder comprising administering to the subject a therapeutically effective amount of an expression vector comprising a transgene comprising human SLC6A1, optionally linked to a promoter sequence that is at least 90%, 95%, or 99% identical to, or comprises, the full length human SLC6A1 native promoter sequence of SEQ ID NO:1 (hNAP).
  • nucleic acids, vectors, or pharmaceutical compositions described herein for use in methods of treating a subject who has a SLC6A1-related disorder.
  • the methods comprise administering to the subject a therapeutically effective amount of a nucleic acid, vector, or pharmaceutical composition as described herein.
  • FIG. 1 Transcriptional regulatory element analysis of the proposed human SLC6A1 native promoter (hNaP). Schematic diagram showing the ⁇ 1.6 kb genomic region of promoter sequence upstream of the SLC6A1 transcriptional start site. Regulatory elements sequence motif search was performed using the Nsite database. The respective locations of these regulatory sites (including the 21-bp motif enhancer element reported previously near -700bp) are listed in this graphical presentation.
  • AAV backbone encompassing essential AAV expression and packaging elements available from Addgene.
  • B The human SLC6A1 native promoter will be subcloned into the AAV backbone to form the pAAV-hNaP intermediate.
  • C Recombinant SLC6A1 gene (coding sequence only) will be further inserted via restriction enzyme digestion and re-ligation. Compatible primer sequences for each cloning step are listed, SEQ ID NOs:4-7.
  • FIGs.3A-C Exemplary sequence of pAAV-hNaP-hSLC6A1 (7375bp total, 4507bp between ITRs; SEQ ID NO:3).
  • the components include: ITR1: CCTGCAGGCAGCTGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACC TTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCC ATCACTAGGGGTTCCT (SEQ ID NO:8) WPRE: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTT GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCT TCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGAC GCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTC GCTTTCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTCCCTTCCTTCCGGGACTTTC G
  • the expression construct is capable of directing expression of the SLC6A1 nucleic acid preferentially in inhibitory neurons and astrocytes.
  • SLC6A1 expression can be driven by a human native SLC6A1 promoter as described herein (e.g., SEQ ID NO:1, or a sequence that is at least 90%, 95%, 97%, or 99% identical to all or part of the full length of SEQ ID NO:1, e.g., and is at least 1100 nucleotides (nt) long, e.g., at least 1200, at least 1300, at least 1369, at least 1400, at least 1500, or all 1521 nt of SEQ ID NO:1), preferably comprising at least nt 175-1320 of SEQ ID NO:1, and preferably comprising at least 10, 20, 30, 50, 75, 100, 150, or all 200 nt of the 3’ end, e.g., of nt 1300-1521, or 1320-1521 (such that the 3’ Attorney
  • the viral vectors e.g., AAV, e.g., packaged in AAV capsids
  • AAV packaged in AAV capsids
  • An exemplary pharmaceutical composition comprising a viral vector, e.g., an AAV, as described herein can include a pharmaceutically acceptable carrier such as balanced saline solution (BSS) and one or more surfactants; exemplary formulations are described in Grossen et al., Eur J Pharm Biopharm.2023 Sep: Attorney Docket No.37314-0124WO1/CMCC 4357 190:1-23.
  • BSS balanced saline solution
  • surfactants exemplary formulations are described in Grossen et al., Eur J Pharm Biopharm.2023 Sep: Attorney Docket No.37314-0124WO1/CMCC 4357 190:1-23.
  • Other pharmaceutical formulation elements known in the art may also be suitable for use in the compositions described herein.
  • the methods include administering a composition comprising a therapeutically effective amount of a nucleic acid encoding GAT1, e.g., human SLC6A1 linked to a human native SLC6A1 promoter, e.g., as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.
  • the methods include administering the composition directly to the brain of the subject.
  • the gene therapy construct can be introduced by catheter (see U.S.
  • the sequence of a protein or nucleic acid (including an ITR, WPRE, or polyA sequence) used in a composition or method described herein is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to an exemplary or reference sequence set forth herein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non- homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For the avoidance of confusion, unless otherwise specified, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol.
  • Biol.48:444-453 algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • hNaP human SLC6A1 native promoter
  • AAV vector was developed by molecular cloning of double strand DNA (ssDNA) fragment encompassing the hNaP sequence flanked by MluI and XbaI sites, as well as another ssDNA fragment encompassing the human SLC6A1 coding sequence flanked by XbaI and EcoRI sites. Sequence integrity of the AAV vector was checked using Sanger Sequencing throughout the whole construct between the two inverted terminal repeats (ITR). See, e.g., FIGs.2A-C and 3A-C. References 1 Goodspeed, K. et al. Current knowledge of SLC6A1-related neurodevelopmental disorders.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Public Health (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Virology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des constructions de thérapie génique SLC6A1, ainsi que des promoteurs SLC6A1, des compositions contenant les constructions et les promoteurs, et leurs procédés d'utilisation.
PCT/US2025/021572 2024-03-26 2025-03-26 Constructions de thérapie génique slc6a1 Pending WO2025207784A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463569989P 2024-03-26 2024-03-26
US63/569,989 2024-03-26

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WO2025207784A1 true WO2025207784A1 (fr) 2025-10-02

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008073303A2 (fr) * 2006-12-07 2008-06-19 Switchgear Genomics Éléments de régulation transcriptionnelle de voies biologiques, outils, et procédés
US20180305689A1 (en) * 2015-04-22 2018-10-25 Mina Therapeutics Limited Sarna compositions and methods of use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008073303A2 (fr) * 2006-12-07 2008-06-19 Switchgear Genomics Éléments de régulation transcriptionnelle de voies biologiques, outils, et procédés
US20180305689A1 (en) * 2015-04-22 2018-10-25 Mina Therapeutics Limited Sarna compositions and methods of use

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