WO2023077143A1 - Compositions utiles pour traiter les troubles dus à la déficience en cdkl5 (cdd) - Google Patents

Compositions utiles pour traiter les troubles dus à la déficience en cdkl5 (cdd) Download PDF

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WO2023077143A1
WO2023077143A1 PCT/US2022/079025 US2022079025W WO2023077143A1 WO 2023077143 A1 WO2023077143 A1 WO 2023077143A1 US 2022079025 W US2022079025 W US 2022079025W WO 2023077143 A1 WO2023077143 A1 WO 2023077143A1
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sequence
cdkl5
hcdkl5
bip
tatk28
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PCT/US2022/079025
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Ralf Schmid
Justin PERCIVAL
Evan Katz
Sean CLARK
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The Trustees Of The University Of Pennsylvania
Amicus Therapeutics, Inc.
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Publication of WO2023077143A1 publication Critical patent/WO2023077143A1/fr

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    • 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
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • 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
    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11022—Cyclin-dependent kinase (2.7.11.22)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • 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

  • CDKL5 Deficiency Disorder is a serious neurodevelopmental disorder affecting young children.
  • the underlying cause is lack of CDKL5 protein expression due to mutations in the X-linked Cyclin- Dependent Kinase-Like 5 gene, CDKL5 (Mendelian Inheritance in Man, MIM: 300203; previously known as STK9), resulting in a range of phenotypes, including EIEE2 (MIM: 300672), a form of early infantile epileptic encephalopathy [Bahi-Buisson, N. et al. Key clinical features to identify girls with CDKL5 mutations.
  • the phenotype may also include a number of other features, such as stereotypic hand movements, severe psychomotor retardation and general hypotonia.
  • the early postnatal onset of symptoms indicates that CDKL5 plays a crucial role in brain development.
  • CDKL5 is also expressed within the mature adult nervous system. CDKL5 is expressed throughout the cell, including the nucleus and the cytoplasm of the cell soma and dendrites.
  • CDKL5 gene mutations are the cause of most cases of CDD, a progressive neurologic developmental disorder and one of the most common causes of cognitive disability in females. Males who have the genetic mutation that causes CDD are affected in devastating ways. Most of them die before birth or in early infancy. See, e.g., ninds.nih.gov/Disorders/Patient-Caregiver-Education/Fact-Sheets/Rett-Syndrome-Fact-Sheet and omim.org/entry/312750.
  • an AAV gene therapy -mediated approach for CDD which provides cross correction in the brain.
  • the gene therapy approach provided herein may reverse post-developmental CDD pathology.
  • a recombinant adeno-associated virus comprises an AAV capsid and a vector genome, wherein the vector genome comprises inverted terminal repeats (ITR) and an expression cassette comprising a nucleic acid sequence comprising coding sequences for BIP-TATk28-hCDKL5 operably linked to regulatory sequences which direct expression thereof, wherein said regulatory sequences comprise a chicken P-actin hybrid promoter and a polyA sequence, and said BIP-TATk28- hCDKL5 is a fusion protein comprising the BIP amino acid sequence of SEQ ID NO: 3, the TATk28-amino acid sequence of SEQ ID NO: 7, and the hCDKL5 amino acid sequence of SEQ ID NO: 24.
  • ITR inverted terminal repeats
  • BIP-TATk28-hCDKL5 is a fusion protein comprising the BIP amino acid sequence of SEQ ID NO: 3, the TATk28-amino acid sequence of SEQ ID NO: 7, and the hCDK
  • the BIP-TATk28-hCDKL5 fusion protein has the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the BIP-TATk28- hCDKL5 fusion protein is encoded by SEQ ID NO: 10 or a sequence 95% identical thereto. In certain embodiments, the chicken P-actin hybrid promoter has a sequence of SEQ ID NO: 12. In certain embodiments, the expression cassette further comprises a Kozak sequence. In certain embodiments, the expression cassette comprises the chicken P-actin hybrid promoter sequence, a Kozak sequence, the BIP-TATk28-hCDKL5 coding sequence, and an SV40 polyA sequence.
  • the expression cassette has the nucleic acid sequence of SEQ ID NO: 13 or sequence at least 95% identical thereto.
  • the vector genome comprises a AAV-5’ ITR (also referenced as 5’ ITR), the chicken P-actin hybrid promoter sequence, a Kozak sequence, the BIP-TATk28-hCDKL5 coding sequence, an SV40 polyA sequence, and a AAV-3’ ITR (also referenced as 3’ ITR).
  • the vector genome has the nucleic acid sequence of SEQ ID NO: 14 or a sequence at least 95% identical thereto.
  • the capsid is an AAVhu68 capsid, an AAVhu95 capsid, or AAVrh91 capsid.
  • a plasmid useful in producing an rAAV particle which comprises an expression cassette comprising a chicken P-actin hybrid promoter sequence, a Kozak sequence, a BIP-TATk28-hCDKL5 coding sequence, and an SV40 polyA sequence is provide.
  • the plasmid contains the vector genome which comprises a 5 ’ ITR, the chicken P-actin hybrid promoter sequence, a Kozak sequence, the BIP- TATk28-hCDKL5 coding sequence, an SV40 polyA sequence, and a 3 ITR.
  • a packaging cell is provided which comprises the expression cassette, vector genome or plasmid.
  • composition comprising a rAAV or a vector as described herein and an aqueous suspension media.
  • a method of treating a subject having CDD, or ameliorating symptoms of CDD, or delaying progression of CDD comprises administrating an effective amount of a rAAV or a vector as described herein to a subject in need thereof.
  • a suspension is formulated for intravenous administration, intrathecal administration, intra-cistema magna administration or intracerebroventricular administration.
  • a rAAV is provided or use in treating a patient having a CDKL5 -deficiency disorder.
  • Use of a rAAV in preparing a medicament for use in treating a patient having a CDKL5-deficiency disorder is provided.
  • FIG 1 is a graphic map of the vector genome in a recombinant AAV particle.
  • the vector genome contains an AAV 5’ inverted terminal repeat (ITR) and an AAV 3’ ITR at the extreme 5’ and 3’ end, respectively.
  • the ITRs flank the sequences of the expression cassette packaged into the AAV capsid which have sequences encoding a BIP-TATk28-hCDKL5 fusion protein, in which BIP refers to a signal peptide and TATk28 is a cell penetrating peptide.
  • the expression cassette further comprises regulatory sequences operably linked to the fusion protein coding sequences, including a hybrid chicken beta actin (CBH) promoter, and an SV40 poly A.
  • CBH hybrid chicken beta actin
  • FIG 2 provides an illustration of cross-correction, in which a secretable BIP- TATk28-hCDKL5 fusion protein is delivered via an AAV which corrects deficient hCDKL5 in vector-transduced cells and delivers secreted hCDKL5 protein to other brain cells.
  • FIG 3A shows western blot analysis of BIP-TATk28-hCDKL5 protein expression in brain of male mice of ID Nos: 734 and 738 (WT PBS), 753 (CDKL5 /y PBS), 783, 855 and 860 (CDKL5 /y BIP.TATk28.hCDKL5), 772 and 832 (CDKL5 /y CDKL5).
  • WT PBS Wi-Fi Protectet analysis of mice of mice of ID Nos: 734 and 738
  • CDKL5 /y PBS 783, 855 and 860
  • CDKL5 /y BIP.TATk28.hCDKL5 772 and 832
  • Male neonatal control or CDKL5 /y mice were injected ICV with PBS or AAVhu68-CBH-BIP-TATk28- hCDKL5 vector or AAVhu68-CBH-hCDKL5 vector.
  • FIG 3B shows western blot analysis of BIP-TATk28-hCDKL5 protein expression in brain of male mice of ID Nos: 1157 and 1162 (WT PBS), 1156 and 1160 (CDKL5 /y PBS), 980 and 1136 (CDKL5 /y BIP.TATk28.hCDKL5), 1202 and 1206 (CDKL5 /y CDKL5).
  • Male P14 control or CDKL5 /y mice were injected ICV with PBS or AAVhu68-CBH-BIP- TATk28-hCDKL5 vector or AAVhu68-CBH-hCDKL5 vector.
  • FIG 3C shows a graph with densitometric quantitation of CDKL5 expression.
  • BIP- TATk28-hCDKL5 protein expression reached 50 percent of wild type (WT) PBS controls and was higher than untagged CDKL5 expression.
  • FIG 3D shows BIP-TATk28_hCDKL5co2 and hCDKL5co2 mRNA expression levels plotted as hCDKL5-co2 transcript per lOOng mRNA.
  • FIG 4 shows representative fluorescent images of sagittal sections of cortex from AAVhu68-CBH-BIP-TATk28-hCDKL5co2 treated CDKL5 /y mice showing the distribution of BIP-TATk28-hCDKL5 protein (yellow) and BIP-TATk28-hCDKL5 mRNA (blue).
  • FIG 5A shows the number of cross corrected cells positive for Cdkl5 protein in neonatal and CDKL5 /y injected mice that were quantitated using Visiopharm® software.
  • FIG 5B shows the number of cells positive for Cdkl5 protein (with or without a signal for Cdkl5 mRNA) in the cortex, striatum, thalamus, hippocampus and hypothalamus of P14 injected control and CDKL5 /y injected mice quantitated using Visiopharm® pathology image analysis software.
  • FIG. 5C further shows the number of cross corrected cells positive for Cdkl5 protein in neonatal and CDKL5 /y injected mice that were quantitated using Visiopharm® software, more specifically in multiple brain regions of P14 BIP-TATk28-hCDKL5 treated CDKL5 /y mice.
  • FIG 6 shows quantitation of brain BIP-TATk28-CDKL5 (BTC) protein expression one month after intravenous injection of PHP.B-CBH-BIP-TATk28-CDKL5 vector in P18 male CDKL5 /y mice.
  • FIG 7 shows that intravenous delivery of PHP.B-CBH-BIP-TATk28-CDKL5 into male P18 CDKL5 /y mice reduced hyperactivity (horizontal activity) measured in open field testing.
  • FIG 8 shows strong expression of BIP-TATk28-CDKL5 (BTC) protein in WT and CDKL5 /y mice at three months after injection in neonatal P0 mice.
  • Figure 8A shows a representative Western blot (upper panel) of BIP-TATk28-CDKL5 protein expression. The lower panel shows the corresponding image of protein loading control using for quantitating BIP-TATk28-CDKL5 protein expression.
  • Figure 8B shows a graph of the densitometric quantitation of BIP-TATk28-CDKL5 protein expression presented as fraction of endogenous wild type CDKL5 expression.
  • FIG 9 shows that PHP.B-CBH-BIP-TATk28-CDKL5 improves hind limb clasping and nest building but not locomotor behavioral deficits in CDKL5 /y mice.
  • PHP.B-CBH-BIP- TATk28-CDKL5 was injected ICV into neonatal mice and tests of behavior were performed three months later.
  • FIG. 9A shows that treatment had no impact on locomotor activity; however, treatment significantly improved hind limb clasping (FIG 9B) and nest building (FIG 9C).
  • FIG 10 shows electroencephalogram (EEG) recording approach in 3-4 and 6-7 month old untreated and treated control and female CDKL5+/- mice.
  • FIG 11 shows the relative power of different brain oscillation frequencies from EEG recordings taken at 12 pm -1 pm during the light cycle in 3-4 month female CDKL5 +/ " mice and wild type (WT) controls.
  • Figure 11A shows perturbation of the relative power of delta, theta alpha bands in CDKL5 +/_ mice.
  • Figure 1 IB shows perturbation of the relative power of the gamma frequency band in CDKL5 +/_ mice.
  • FIG 12 shows the relative power of different brain oscillation frequencies recorded by EEG at 12 am -1 am during the night cycle in 3-4 month female CDKL5 +/ " mice and wild type (WT) controls.
  • Figure 12A shows perturbation of the relative power of delta, theta alpha frequency bands in CDKL5 +/_ mice.
  • Figure 12B shows perturbation of the relative power of the gamma frequency band in CDKL5 +/_ mice.
  • FIG 13A and 13B show that BIP- TATk28k-hCDKL5 reduces aberrant delta/theta and gamma power in CDKL5 +/_ mice during the middle of the light cycle (12pm - 2pm window).
  • FIG 7A shows the fraction of total power output at delta (0-4 Hz), theta (4-8 Hz), alpha (8-13 Hz) and beta (13-24 Hz) frequency bands.
  • FIG 7B shows the fraction of total pro-seizure gamma (15-50 Hz) power output.
  • FIGs 14A and 14B show that BIP-TATk28k-hCDKL5 reduces abnormalities in delta, alpha, and gamma frequency bands in CDKL5 +/_ mice at the dark to light transition (6am-7am window).
  • FIG 14A shows the fraction of total power output at delta (0-4 Hz), theta (4-8 Hz), alpha (8-13 Hz) and beta (13-24 Hz) frequency bands. The fraction of delta, theta power was decreased in PBS treated CDKL5 +/_ mice compared with PBS treated wild type controls.
  • FIG 14B shows the fraction of total pro-seizure gamma (15-50 Hz) power output.
  • FIGs 15A and 15B show that BIP-TATk28k-hCDKL5 reduces abnormalities in theta, alpha, and gamma frequencies in CDKL5 +/_ mice during the light to dark transition (6pm-7pm window).
  • FIG 15A shows the fraction of total power output at delta (0-4 Hz), theta (4-8 Hz), alpha (8-13 Hz) and beta (13-24 Hz) frequency bands.
  • FIG 15B shows the fraction of gamma (15-50 Hz) power output.
  • FIG 16A shows that BIP-TATk28k-hCDKL5 reduces abnormal gamma frequency power in aged female CDKL5 +/_ mice during the middle of the light cycle (6am-7am).
  • FIG 16B shows that BIP-TATk28k-hCDKL5 reduces abnormal gamma frequency power in aged female CDKL5 +/_ mice during the middle of the light cycle (12pm - 2pm).
  • FIGs 16C to 16F show that BIP-TATk28k-hCDKL5 reduces abnormalities in gamma frequency power in CDKL5 +/_ mice at the dark to light transition (6am-7am window).
  • FIG 16C shows a representative 1-hour long electrocardiogram traces from WT PBS treated mice.
  • FIG 16D shows a representative 1-hour long electrocardiogram traces from CDKL5 +/_ mice PBS treated mice.
  • FIG. 16E shows a representative 1-hour long electrocardiogram traces from WT BIP-TATk28-hCDKL5 mice.
  • FIG. 16F shows a representative 1-hour long electrocardiogram traces from CDKL5 +/ " BIP-TATk28-hCDKL5 treated mice.
  • FIG. 17A further shows cortical protein expression (CDKL5) in mice, plotted as percent wild-type-PBS expression in CDKL5 /y with P14 treatment.
  • FIG. 17B further shows cortical mRNA expression, more specifically, BIP-TATk28-CDKL5 (BTC) and hCDKL5 mRNA expression levels plotted as hCDKL5-co2 transcript per lOOng mRNA.
  • BTC BIP-TATk28-CDKL5
  • hCDKL5 mRNA expression levels plotted as hCDKL5-co2 transcript per lOOng mRNA.
  • FIGs. 18A to 18F further shows representative fluorescent images (Immunofluorescence and In Situ Hybridization) of cortex from AAVhu68-CBH-BIP- TATk28-hCDKL5co2 treated CDKL5 /y mice showing the distribution of BIP-TATk28- hCDKL5 protein (yellow) and BIP-TATk28-hCDKL5 mRNA (blue). Nuclei (purple) are counterstained with DAPI (4',6-diamidino-2-phenylindole).
  • FIG. 19A shows a representative microscopy image of CDKL5 expression in hippocampal cross-section tissue of female control CDKL5 +/_ mice administered with PBS.
  • FIG. 19B shows a representative microscopy image of CDKL5 expression in hippocampal cross-section tissue of female control CDKL5 +/_ mice treated with AAV.BIP-TATk28- hCDKL5.
  • FIG. 19C shows hippocampal BIP-TATk28-hCDKL5 expression, plotted as percent wildtype-PBS expression.
  • FIG. 19D shows western blot analysis of BIP-TATk28- hCDKL5 and endogenous CDKL5 protein expression (in comparison with the total protein control) in hippocampal tissue of female CDKL5 +/_ mice.
  • FIG. 20 shows biodistribution results for peripheral tissue, brain, dorsal root ganglion (DRG), spinal cord (SC), plotted as GC/diploid cell.
  • DRG dorsal root ganglion
  • SC spinal cord
  • FIG. 21 shows RNA biodistribution for select tissues of peripheral organs, brain, DRG and SC. The values are summarized in tables 2A, 2B, and 2C below.
  • FIG. 22 shows summarized results of the ELISPOT analysis (Tables 3A and 3B) for AAVhu68, plotted as spot forming units (SFU) per 10 6 (million) cells.
  • FIG. 23 shows summarized results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5, plotted as SFU per 10 6 (million) cells. These results indicate some liver transduction with AAVhu68 and hCDKL5 expression following ICM administration.
  • FIG. 24A shows results of the ELISPOT analysis (Table 3 A) for AAVhu68 pool A, plotted as SFU per 10 6 (million) cells.
  • FIG. 24B shows results of the ELISPOT analysis (Table 3A) for AAVhu68 pool B, plotted as SFU per 10 6 (million) cells.
  • FIG. 24C shows results of the ELISPOT analysis (Table 3A) for AAVhu68 pool C, plotted as SFU per 106 (million) cells.
  • FIG. 3 A shows results of the ELISPOT analysis for AAVhu68 pool A, plotted as SFU per 10 6 (million) cells.
  • FIG. 24D shows results of the ELISPOT analysis (Table 3 A) for AAVhu68 baseline PBMC (for subjects 171164, 191410, HS1602026 as analyzed with dimethyl sulfoxide (DMSO), Pool A, Pool B, and Pool C), plotted as SFU per 106 (million) cells.
  • FIG. 24E shows results of the ELISPOT analysis (Table 3A) for AAVhu68 D35/36 PBMC (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A, Pool B, and Pool C), plotted as SFU per 10 6 (million) cells.
  • FIG. 3 A shows results of the ELISPOT analysis for AAVhu68 baseline PBMC (for subjects 171164, 191410, HS1602026 as analyzed with dimethyl sulfoxide (DMSO), Pool A, Pool B, and Pool C), plotted as SFU per 106 (million) cells.
  • FIG. 24E shows results of the ELISPOT analysis (Table 3A
  • FIG. 24F shows results of the ELISPOT analysis (Table 3A) for AAVhu68 spleen sample (for subjects 171164, 191410, HS1602026 as analyzed with DMSO, Pool A, Pool B, and Pool C), plotted as SFU per 10 6 (million) cells.
  • FIG. 24G shows results of the ELISPOT analysis (Table 3A) for AAVhu68 liver sample (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A, Pool B, and Pool C), plotted as SFU per 10 6 (million) cells.
  • FIG. 3A shows results of the ELISPOT analysis for AAVhu68 spleen sample (for subjects 171164, 191410, HS1602026 as analyzed with DMSO, Pool A, Pool B, and Pool C), plotted as SFU per 10 6 (million) cells.
  • 24H shows results of the ELISPOT analysis (Table 3A) for AAVhu68 lymph node sample (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A, Pool B, and Pool C), plotted as SFU per 10 6 (million) cells.
  • FIG. 25 A shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool A-l, plotted as SFU per 10 6 (million) cells.
  • FIG. 25B shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool A, plotted as SFU per 10 6 (million) cells.
  • FIG. 25C shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool B, plotted as SFU per 10 6 (million) cells.
  • FIG. 25 A shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool A-l, plotted as SFU per 10 6 (million) cells.
  • FIG. 25B shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool A-l, plotted as SFU per 10 6 (million) cells.
  • 25D shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool C, plotted as SFU per 10 6 (million) cells.
  • FIG. 25E shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool D, plotted as SFU per 10 6 (million) cells.
  • FIG. 25F shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 baseline PBMC sample (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool B, Pool C, Pool D peptides), plotted as SFU per 10 6 (million) cells.
  • 25 G shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 D35/D36 PBMC sample (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool B, Pool C, Pool D peptides), plotted as SFU per 10 6 (million) cells.
  • FIG. 25H shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 spleen sample (for subjects 171164, 191410, HS1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool B, Pool C, Pool D peptides), plotted as SFU per 10 6 (million) cells.
  • FIG. 3A and 3B shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 spleen sample (for subjects 171164, 191410, HS1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool
  • FIG. 251 shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 liver sample (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool B, Pool C, Pool D peptides), plotted as SFU per 10 6 (million) cells.
  • FIG. 25J shows results of the ELISPOT analysis (Tables 3 A and 3B) for hCDKL5 lymph node sample (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool B, Pool C, Pool D peptides), plotted as SFU per 10 6 (million) cells. Overall, these results indicate a transgene specific T cell responses seen in all animals.
  • FIG. 26A shows measured velocity in left medial nerve.
  • FIG. 26B shows measured velocity in right medial nerve.
  • FIG. 26C shows NP amplitude in left medial nerve.
  • FIG. 26D shows NP amplitude in right medial nerve.
  • FIG. 26E shows peak to peak (PP) amplitude in left medial nerve.
  • FIG. 26F shows PP amplitude in right medial nerve.
  • FIG. 26G shows measured velocity in left sural nerve.
  • FIG. 26H shows measured velocity in right sural nerve.
  • FIG. 261 shows NP amplitude in left sural nerve.
  • FIG. 26J shows NP amplitude in right sural nerve.
  • FIG. 26K shows PP amplitude in left sural nerve.
  • FIG. 26L shows PP amplitude in right sural nerve.
  • compositions and methods for treating CDD are provided herein.
  • the secretable CDKL5 lusion proteins delivered via the AAV provided herein are believed to provide broader brain CDKL5 delivery and expression, contributing to crosscorrection (i.e., correction in cells not initially transduced with the rAAV vector).
  • the various nucleic acid sequences provided herein are useful for packaging BIP- TATk28-hCDKL5 coding sequence into a suitable vector (e.g., a rAAV) or a genetic element useful for manufacture (e.g., a plasmid).
  • a suitable vector e.g., a rAAV
  • a genetic element useful for manufacture e.g., a plasmid
  • the hCDKL5 sequences herein encode human CDKL5 protein isoform 1.
  • the wildtype hCDKL5 sequences are reproduced in SEQ ID NO: 1.
  • the hCDKL5 sequence is fused at its N-terminus to an exogenous BIP leader sequence (Bip) and a peptide uptake enhancer (TATk28), which is designed to enhance uptake of the peptide by cells following expression of the fusion protein from cells transduced with the expression cassette.
  • the fusion protein contains the human CDKL5 amino acid sequence of SEQ ID NO: 24.
  • a sequence having at least 95% to 100% identity to SEQ ID NO: 24 (also amino acid 61 to 1020 of SEQ ID NO: 11) and/or values therebetween, at least 96% identity to 100% identity, at least 97% identity, at least 98% identity, at least 99% identity, 99% to 100% identity to SEQ ID NO: 24 may be used which provides functional hCDKL5 activity.
  • the hCDKL5 coding sequences provided herein are engineered sequences.
  • SEQ ID NO: 2 provides engineered sequences which are referred to interchangeably as hCDKL5co2 or hCDKL5 engineered sequences.
  • CDKL5 coding sequences which are 95% to 100% identical to the contiguous sequence of SEQ ID NO; 5 which encode SEQ ID NO: 1 (optionally lacking the initial Met, such as in SEQ ID NO: 24), i.e., at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 1, or up to 100% identical, and values therebetween which encode SEQ ID NO: 1 (optionally lacking the initial Met, as in SEQ ID NO: 24).
  • the CDKL5 fusion protein provided herein further comprises an uptake peptide fused directly or via a short linker (e.g., GGGGS) to the N-terminus of the hCDKL5 protein.
  • the uptake peptide is TATk28-peptide (also referenced to as the TAT peptide) having the amino acid sequence of SEQ ID NO: 7.
  • the TATk28-peptide is encoded by SEQ ID NO: 5 or a sequence at least 99% identical thereto which encodes SEQ ID NO: 7.
  • the CDKL5 fusion protein provided herein further comprises a BIP signal peptide (also referenced to as Bip, or BiP signal peptide) fused to the N-terminus of the TATk28- optional linker-CDKL5 fusion.
  • the signal peptide has the sequence of SEQ ID NO: 3.
  • the BIP signal peptide is encoded by SEQ ID NO: 4.
  • the BIP signal peptide is encoded by a sequence at least 99% identical to SEQ ID NO: 4 which encodes SEQ ID NO: 4.
  • the BIP-TATk28-hCDKL5 is a fusion protein having the amino acid sequence of SEQ ID NO: 11. Throughout the specification, this is also used interchangeably with BIP-TAT-hCDKL5 or BIP-TATk28-hCDKL5-co2. In certain embodiments, the fusion protein has a sequence at least 99% identical to SEQ ID NO: 11.
  • the BIP-TATk28-hCDKL5 is encoded by a nucleic acid sequence of SEQ ID NO: 10 or a sequence at least 95% to 100% identical to SEQ ID NO: 10, and/or values therebetween, at least 96% identity to 100% identity, at least 97% identity, at least 98% identity, at least 99% identity, 99% to 100% identity to SEQ ID NO: 10.
  • the CDKL5 is made within cell following administration with expression cassette or a vector genome comprising BIP-TATk28-hCDKL5, wherein some expressed hCDKL5 stays within a cell, while some is secreted and taken up by neighboring cells.
  • This approach allows for cross correction (i.e., delivery of hCDKL5 to cells not transduced by the AAV.BIP-TATk28-hCDKL5.
  • cross correction i.e., delivery of hCDKL5 to cells not transduced by the AAV.BIP-TATk28-hCDKL5.
  • an early study shows cross-correction in hippocampus, cortex, striatum, thalamus, and hypothalamus. See, Example 2 below.
  • the extent of cross correction is examined using visualization of number of cells positive for Cdkl5 protein (cross corrected and transduced cells) in comparison with or without a signal for Cdkl5 mRNA (transduced cells) in various tissues.
  • a gene therapy vector comprises an expression cassette comprising a nucleic acid sequence comprising coding sequences for BIP-TATk28-hCDKL5 operably linked to regulatory sequences which direct expression thereof.
  • an “expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein (i.e., a CDKL5 coding sequence), enzyme or other useful gene product, mRNA, etc.) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence and its gene product.
  • a biologically useful nucleic acid sequence e.g., a gene cDNA encoding a protein (i.e., a CDKL5 coding sequence), enzyme or other useful gene product, mRNA, etc.
  • regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence and its gene product.
  • operably linked sequences include both regulatory sequences that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in trans or cis nucleic acid sequence.
  • Such regulatory sequences typically include, e.g., one or more of a promoter(s), an enhancer(s), an intron(s), a Kozak sequence, a polyadenylation sequence, and a TATA signal.
  • the expression cassette may contain regulatory sequences upstream (5’ to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3 ’ to) a gene sequence, e.g., 3’ untranslated region (3’ UTR) comprising a polyadenylation site, among other elements.
  • the regulatory sequences are operably linked to the nucleic acid sequence of a gene product, wherein the regulatory sequences are separated from nucleic acid sequence of a gene product by an intervening nucleic acid sequences, i.e., 5 ’-untranslated regions (5’UTR).
  • the expression cassette comprises nucleic acid sequence of one or more of gene products.
  • the expression cassette can be a monocistronic or a bicistronic expression cassette.
  • the term “transgene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell.
  • such an expression cassette can be used for generating a viral vector and contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.
  • a vector genome may contain two or more expression cassettes.
  • an expression cassette comprising the coding sequence for BIP-TATk28-hCDKL5 operably linked to regulatory sequences for directed its expression.
  • the regulatory sequences comprise a chicken P-actin hybrid (also referenced to as CBh or CBH) promoter and a polyA sequence.
  • the chicken P-actin hybrid promoter has a sequence of SEQ ID NO: 12.
  • the expression cassette further comprises a Kozak sequence.
  • the expression cassette comprises the chicken P-actin hybrid promoter sequence, a Kozak sequence, the BIP-TATk28-hCDKL5 coding sequence, and an SV40 polyA sequence.
  • the SV40 polyA sequence has a sequence of SEQ ID NO: 26.
  • the expression cassette has a nucleic acid sequence of SEQ ID NO: 13 or sequence at least 95% identical thereto, or at least 96% identical thereto, or at least 97% identical thereto, or at least 98% identical thereto, or at least 99% identical, or 95% to 100% identical to SEQ ID NO: 13, and/or values therebetween.
  • such an expression cassette to be packed into a viral vector contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.
  • the vector genome comprises a 5’ ITR, the chicken P-actin hybrid promoter sequence, a Kozak sequence, the BIP-TATk28-hCDKL5 coding sequence, an SV40 polyA sequence, and a 3’ ITR.
  • the vector genome has the nucleic acid sequence of SEQ ID NO: 14 or a sequence at least 95% identical thereto, or at least 96% identical thereto, or at least 97% identical thereto, or at least 98% identical thereto, or at least 99% identical, or 95% to 100% identical to SEQ ID NO: 14, and/or values therebetween.
  • the target cell may be a central nervous system cell.
  • the target cell is one or more of an excitatory neuron, an inhibitory neuron, a glial cell, a cortex cell, a frontal cortex cell, a cerebral cortex cell, a spinal cord cell.
  • the target cell is a peripheral nervous system (PNS) cell, for example a retina cell.
  • PNS peripheral nervous system
  • Other cells other than those from nervous system may also be chosen as a target cell, such as a monocyte, a B lymphocyte, a T lymphocyte, a NK cell, a lymph node cell, a tonsil cell, a bone marrow mesenchymal cell, a stem cell, a bone marrow stem cell, a heart cell, an epithelium cell, a esophagus cell, a stomach cell, a fetal cut cell, a colon cell, a rectum cell, a liver cell, a kidney cell, a lung cell, a salivary gland cell, a thyroid cell, an adrenal cell, a breast cell, a pancreas cell, an islet of Langerhans cell, a gallbladder cell, a prostate cell, a urinary bladder cell, a skin cell, a uterus cell, a cervix cell, a testis cell, or any other cell which expresses a functional CDKL5 protein in a subject without CDD.
  • an additional or alternative promoter sequence may be included as part of the expression control sequences (regulatory sequences), e.g., located between the selected 5’ ITR sequence and the coding sequence.
  • Constitutive promoters, regulatable promoters [see, e.g., WO 2011/126808 and WO 2013/04943], tissue specific promoters, or a promoter responsive to physiologic cues may be utilized in the vectors described herein.
  • the promoter(s) can be selected from different sources, e.g., human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polymovirus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters, herpes simplex virus (HSV-1) latency associated promoter (LAP), rouse sarcoma virus (RSV) long terminal repeat (LTR) promoter, neuronspecific promoter (RNSE), platelet derived growth factor (PDGF) promoter, hSYN, melaninconcentrating hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP), and the chicken beta-actin promoter.
  • CMV human cytomegalovirus
  • MBP myelin basic protein
  • GFAP glial fibrillary acidic protein
  • HSV-1 herpes simplex virus
  • LAP rouse sar
  • a vector may contain one or more other appropriate transcription initiation sequences, transcription termination sequences, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA for example WPRE; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • a suitable enhancer is the CMV enhancer.
  • Other suitable enhancers include those that are appropriate for desired target tissue indications.
  • the regulatory sequences comprise one or more expression enhancers.
  • the regulatory sequences contain two or more expression enhancers.
  • an enhancer may include a CMV immediate early (CMV IE) enhancer.
  • CMV IE CMV immediate early
  • the expression cassette further contains an intron, e.g., the chicken beta-actin intron.
  • the intron is a chimeric intron (CI) - a hybrid intron consisting of a human beta-globin splice donor and immunoglobulin G (IgG) splice acceptor elements.
  • suitable introns include those known in the art, e.g.
  • polyA sequences include, e.g., Rabbit globin poly A, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyAs.
  • bGH bovine growth hormone
  • synthetic polyAs one or more sequences may be selected to stabilize mRNA.
  • the expression cassettes provided may include one or more expression enhancers such as post-transcriptional regulatory element from hepatitis viruses of woodchuck (WPRE), human (HPRE), ground squirrel (GPRE) or arctic ground squirrel (AGSPRE); or a synthetic post-transcriptional regulatory element. These expressionenhancing elements are particularly advantageous when placed in a 3' UTR and can significantly increase mRNA stability and/or protein yield.
  • the expressions cassettes provided include a regulator sequence that is a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a variant thereof.
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • Suitable WPRE sequences are provided in the vector genomes described herein and are known in the art (e.g., such as those are described in US Patent Nos. 6,136,597, 6,287,814, and 7,419,829, which are incorporated by reference).
  • the WPRE is a variant that has been mutated to eliminate expression of the woodchuck hepatitis B virus X (WHX) protein, including, for example, mutations in the start codon of the WHX gene (See, Zanta- Boussif et al., Gene Ther. 2009 May;16(5):605-19, which is incorporated by reference).
  • enhancers are selected from a non-viral source.
  • no WPRE sequence is present.
  • a recombinant adeno-associated virus useful for treating CDD.
  • the rAAV comprises (a) an AAV capsid; and (b) a vector genome packaged in the AAV capsid of (a).
  • the AAV capsid selected targets the cells to be treated.
  • the capsid is from Clade F.
  • the Clade F AAV capsid is selected from an AAVhu68 capsid [See, e.g., US2020/0056159; PCT/US21/55436; SEQ ID NO: 19 and 21 for nucleic acid sequence; SEQ ID NO: 20 for amino acid sequence] or an AAVhu95 capsid [See, e.g., US Provisional Application No. 63/251,599, filed October 2, 2201; SEQ ID NO: 22 (hu95 nucleic acid sequence) and SEQ ID NO: 23 (hu95 amino acid sequence).
  • the AAV capsid is a Clade A capsid, such as AAVrh91 capsid (nucleic acid sequence of SEQ ID NOs: 15 and 17). See, PCT7US20/030266, filed April 29, 2020, now published WO2020/223231, which is incorporated by reference herein and International Application No. PCT/US21/45945, filed August 13, 2021 which are incorporated herein by reference.
  • the AAV capsid for the compositions and methods described herein is chosen based on the target cell.
  • the AAV capsid transduces a CNS cell and/or a PNS cell.
  • other AAV capsid may be chosen, the AAV capsid is selected from a cy02 capsid, a rh43 capsid, an AAV8 capsid, a rhOl capsid, an AAV9 capsid, an rh8 capsid, a rhlO capsid, a bbOl capsid, a hu37 capsid, a rh02 capsid, a rh20 capsid, a rh39 capsid, a rh64 capsid, an AAV6 capsid, an AAV1 capsid, a hu44 capsid, a hu48 capsid, a cy05 capsid a
  • the AAV capsid is a Clade F capsid, such as AAV9 capsid, AAVhu68 capsid, hu31 capsid, hu32 capsid, or a variation thereof. See, e.g., WO 2005/033321 published April 14, 2015, WO 2018/160582, and US 2015/0079038, each of which is incorporated herein by reference in its entirety.
  • the AAV capsid is a non-clade F capsid, for example a Clade A, B, C, D, or E capsid.
  • the non-Clade F capsid is an AAV 1 or a variation thereof.
  • the AAV capsid transduces a target cell other than the nervous system cells.
  • the AAV capsid is a Clade A capsid (e.g., AAV1, AAV6, AAVrh91), a Clade B capsid (e.g., AAV 2), a Clade C capsid (e.g., hu53), a Clade D capsid (e.g., AAV7), or a Clade E capsid (e.g., rhlO).
  • the term “clade” as it relates to groups of AAV refers to a group of AAV which are phylogenetically related to one another as determined using a Neighbor- Joining algorithm by a bootstrap value of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no more than 0.05, based on alignment of the AAV vpl amino acid sequence.
  • the Neighbor-Joining algorithm has been described in the literature. See, e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Computer programs are available that can be used to implement this algorithm. For example, the MEGA v2.
  • a rAAV is composed of an AAV capsid and a vector genome.
  • An AAV capsid is an assembly of a heterogeneous population of vpl, a heterogeneous population of vp2, and a heterogeneous population of vp3 proteins.
  • the term “heterogeneous” or any grammatical variation thereof refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences.
  • heterogeneous refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences.
  • heterogeneous population refers to differences in the amino acid sequence of the vpl, vp2 and vp3 proteins within a capsid.
  • the AAV capsid contains subpopulations within the vp 1 proteins, within the vp2 proteins and within the vp3 proteins which have modifications from the predicted amino acid residues. These subpopulations include, at a minimum, certain deamidated asparagine (N or Asn) residues.
  • certain subpopulations comprise at least one, two, three or four highly deamidated asparagines (N) positions in asparagine - glycine pairs and optionally further comprising other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications.
  • AAV capsids are provided which have a heterogeneous population of AAV capsid isoforms (i.e., VP1, VP2, VP3) which contain multiple highly deamidated “NG” positions.
  • the highly deamidated positions are in the locations identified below, with reference to the predicted full-length VP 1 amino acid sequence.
  • the capsid gene is modified such that the referenced “NG” is ablated and a mutant “NG” is engineered into another position.
  • the AAV capsid is a AAVhu68 capsid, or an AAVrh91 capsid.
  • the AAVhu68 capsid comprises amino acid sequence of SEQ ID NO: 20.
  • the AAVhu68 capsid comprises: (i) AAVhu68 vpl proteins, AAVhu68 vp2 proteins, and AAVhu68 vp3 proteins produced from a nucleic acid sequence encoding SEQ ID NO: 20; or (ii) heterogenous populations of AAVhu68 vpl, AAVhu68 vp2 and AAVhu68 vp3 proteins, wherein the subpopulations of the AAVhu68 vpl, AAVhu68 vp2 and AAV hu68 vp3 proteins comprise at least 50% to 100% deamidated asparagines (N) in asparagine - glycine pairs at each of positions 57, 329, 452, 512,
  • the nucleic acid sequence encoding AAVhu68 vpl protein is SEQ ID NO: 19, or a sequence at least 80% to at least 99% identical to SEQ ID NO: 19 which encodes the amino acid sequence of SEQ ID NO: 20; optionally wherein the nucleic acid sequence is at least 80% to 97% identical to SEQ ID NO: 19.
  • the nucleic acid sequence encoding AAVhu68 vpl protein is SEQ ID NO: 21, or a sequence at least 80% to at least 99% identical to SEQ ID NO: 21 which encodes the amino acid sequence of SEQ ID NO: 20; optionally wherein the nucleic acid sequence is at least 80% to 97% identical to SEQ ID NO: 21.
  • target cell and “target tissue” can refer to any cell or tissue which is intended to be transduced by the subject AAV vector.
  • the term may refer to any one or more of muscle, liver, lung, airway epithelium, central nervous system, neurons, eye (ocular cells), or heart.
  • a “vector genome” refers to the nucleic acid sequence packaged inside the rAAV capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • a vector genome contains, at a minimum, from 5’ to 3’, an AAV 5’ ITR, coding sequence(s) (i.e., transgene(s)), and an AAV3’ ITR.
  • the ITRs are from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected.
  • the ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV.
  • ITRs e.g., self-complementary (scAAV) ITRs
  • scAAV self-complementary
  • Both single-stranded AAV and self- complementary (sc) AAV are encompassed with the rAAV.
  • a shortened version of the 5’ ITR termed AITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted.
  • the vector genome includes a shortened AAV2 ITR of 130 base pairs, wherein the external A elements is deleted.
  • the shortened ITR reverts back to the wild-type (WT) length of 145 base pairs during vector DNA amplification using the internal (A’) element as a template.
  • WT wild-type
  • AAV 5’ and 3’ ITRs are used.
  • the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped.
  • other configurations of these elements may be suitable.
  • the transgene is a nucleic acid coding sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest.
  • the nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a cell of a target tissue. Suitable components of a vector genome are discussed in more detail herein.
  • a “vector genome” contains, at a minimum, from 5’ to 3’, a vector-specific sequence, a nucleic acid sequence encoding protein of interest operably linked to regulatory control sequences (which direct their expression in a target cell), where the vector-specific sequence may be a terminal repeat sequence which specifically packages the vector genome into a viral vector capsid or envelope protein.
  • the vector-specific sequence may be a terminal repeat sequence which specifically packages the vector genome into a viral vector capsid or envelope protein.
  • AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.
  • the vector genome is an expression cassette having inverted terminal repeat (ITR) sequences necessary for packaging the vector genome into the AAV capsid at the extreme 5 ’ and 3 ’ end and containing therebetween a CDLK5 gene as described herein operably linked to sequences which direct expression thereof.
  • ITR inverted terminal repeat
  • the term “host cell” may refer to the packaging cell line in which the rAAV is produced from the plasmid. In the alternative, the term “host cell” may refer to the target cell in which expression of the transgene is desired.
  • an rAAV production system useful for producing a rAAV as described herein.
  • the production system comprises a cell culture comprising (a) a nucleic acid sequence encoding an AAV capsid protein; (b) the vector genome; and (c) sufficient AAV rep functions and helper functions to permit packaging of the vector genome into the AAV capsid.
  • the vector genome comprises a AAV 5’ ITR, expression cassette comprising engineered hCDKL5 coding sequence, optionally further comprising BiP and or Tatk28 peptides, operably linked to expression control sequences which control expression thereof, and AAV 3 ’ ITR.
  • the vector genome comprises a AAV 5’ ITR, expression cassette comprising BiP peptide, Tatk28, and engineered hCDKL5 coding sequence operably linked to expression control sequences which control expression thereof, and AAV 3’ ITR.
  • the vector genome comprises a AAV 5 ’ ITR, expression cassette comprising nucleic acid sequence of SEQ ID NO: 13, and AAV 3’ ITR.
  • the vector genome is SEQ ID NO: 14.
  • the cell culture is a human embryonic kidney 293 cell culture.
  • the AAV rep is from a different AAV.
  • wherein the AAV rep is from AAV2.
  • the AAV rep coding sequence and cap genes are on the same nucleic acid molecule, wherein there is optionally a spacer between the rep sequence and cap gene.
  • the vector genomes can be carried on any suitable vector, e.g., a plasmid, which is delivered to a packaging host cell.
  • a suitable vector e.g., a plasmid
  • the plasmids useful in this invention may be engineered such that they are suitable for replication and packaging in vitro in prokaryotic cells, insect cells, mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and/or can be readily designed by one of skill in the art.
  • a plasmid useful in producing an rAAV particle which comprises an expression cassette comprising a chicken P-actin hybrid promoter sequence, a Kozak sequence, a BIP-TATk28-hCDKL5 coding sequence, and an SV40 polyA sequence, said BIP-TATk28-hCDKL5 encoding a protein comprising the BIP amino acid sequence of SEQ ID NO: 3, the TATk28-amino acid sequence of SEQ ID NO: 7, and the hCDKL5 amino acid sequence of SEQ ID NO: 24.
  • the expression cassette has the nucleic acid sequence of SEQ ID NO: 13 or sequence at least 95% identical thereto.
  • the expression cassette has the nucleic acid sequence of SEQ ID NO: 25 or sequence at least 95% identical thereto.
  • the vector genome has the nucleic acid sequence of SEQ ID NO: 14 or a sequence at least 95% identical thereto.
  • a gene therapy vector refers to a rAAV as described herein, which is suitable for use in treating a patient.
  • the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the gene.
  • the cap and rep genes can be supplied in trans.
  • the manufacturing process for rAAV involves method as described in US Provisional Patent Application No. 63/371,597, filed August 16, 2022, and US Provisional Patent Application No. 63/371,592, filed August 16, 2022, which are incorporated herein by reference in its entirety.
  • the selected genetic element may be delivered to an AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • Stable AAV packaging cells can also be made.
  • the methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).
  • AAV intermediate or “AAV vector intermediate” refers to an assembled rAAV capsid which lacks the desired genomic sequences packaged therein. These may also be termed an “empty” capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are nonfunctional to transfer the gene of interest to a host cell.
  • the recombinant adeno-associated virus (AAV) described herein may be generated using techniques which are known. See, e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2.
  • Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; an expression cassette composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein.
  • ITRs AAV inverted terminal repeats
  • a production cell culture useful for producing a recombinant AAV having a capsid selected from AAVhu68, AAVrh91, or hu95 contains a nucleic acid which expresses the AAVhu68 capsid protein in the host cell (e.g., SEQ ID NO: 19 or SEQ ID NO: 21; a nucleic acid molecule suitable for packaging into the AAVhu68 capsid, e.g., a vector genome which contains AAV ITRs and a non-AAV nucleic acid sequence encoding a gene operably linked to regulatory sequences which direct expression of the gene in a host cell; and sufficient AAV rep functions and adenovirus helper functions to permit packaging of the vector genome into the recombinant AAVhu68, or AAVrh91 capsid (e.g., SEQ ID NO: 15 or SEQ ID NO: 17), AAVhu95 capsid (e.g., SEQ
  • the cell culture is composed of mammalian cells (e.g., human embryonic kidney 293 cells, among others) or insect cells (e.g., Spodoptera frugiperda (Sf9) cells).
  • mammalian cells e.g., human embryonic kidney 293 cells, among others
  • insect cells e.g., Spodoptera frugiperda (Sf9) cells.
  • baculovirus provides the helper functions necessary for packaging the vector genome into the recombinant AAVhu68, AAVrh91, or AAVhu95 capsid.
  • rep functions are provided by an AAV other than AAV2, selected to complement the source of the ITRs.
  • cells are manufactured in a suitable cell culture (e.g., HEK 293 or Sf9) or suspension.
  • Methods for manufacturing the gene therapy vectors described herein include methods well known in the art such as generation of plasmid DNA used for production of the gene therapy vectors, generation of the vectors, and purification of the vectors.
  • the gene therapy vector is an AAV vector and the plasmids generated are an AAV cis-plasmid encoding the AAV vector genome and the gene of interest, an AAV trans-plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid.
  • the vector generation process can include method steps such as initiation of cell culture, passage of cells, seeding of cells, transfection of cells with the plasmid DNA, post- transfection medium exchange to serum free medium, and the harvest of vector-containing cells and culture media.
  • the harvested vector-containing cells and culture media are referred to herein as crude cell harvest.
  • the gene therapy vectors are introduced into insect cells by infection with baculovirus-based vectors.
  • Zhang et al., 2009 Adenovirus-adeno- associated virus hybrid for large-scale recombinant adeno-associated virus production, Human Gene Therapy 20:922-929, the contents of each of which is incorporated herein by reference in its entirety.
  • the crude cell harvest may thereafter be subject to further processing including, concentration of the vector harvest, diafdtration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, fdtration of microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and/or formulation and filtration to prepare bulk vector.
  • An affinity chromatography purification followed anion exchange resin chromatography are used to purify the vector drug product and to remove empty capsids.
  • GC genome copies
  • the number of particles (pt) per 20 pL loaded is then multiplied by 50 to give particles (pt) /mL.
  • Pt/mL divided by GC/mL gives the ratio of particles to genome copies (pt/GC).
  • Pt/mL-GC/mL gives empty pt/mL.
  • Empty pt/mL divided by pt/mL and x 100 gives the percentage of empty particles.
  • the methods include subjecting the treated AAV stock to SDS -polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon.
  • Anti-AAV capsid antibodies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably the Bl anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281- 9293).
  • a secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti- IgG antibody containing a detection molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase.
  • a method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit.
  • a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit.
  • samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent (e.g., DTT), and capsid proteins were resolved on pre-cast gradient polyacrylamide gels (e.g., Novex).
  • Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining method, i.e., SYPRO ruby or coomassie stains.
  • the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR).
  • Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using primers and a TaqManTM Anorogenic probe specific for the DNA sequence between the primers. The number of cycles required to reach a defined level of Auorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.
  • DNase I or another
  • an optimized q-PCR method which utilizes a broad spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal to the sample size.
  • the proteinase K buffer may be concentrated to 2-fold or higher. Typically, proteinase K treatment is about 0.2 mg/mL, but may be varied from 0. 1 mg/mL to about 1 mg/mL.
  • the treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g., up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes).
  • heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e.g., about 70 to about 90 °C) and the time extended (e.g., about 20 minutes to about 30 minutes). Samples are then diluted (e.g., 1000- fold) and subjected to TaqMan analysis as described in the standard assay.
  • droplet digital PCR may be used.
  • ddPCR droplet digital PCR
  • methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. doi: 10. 1089/hgtb.2013. 131. Epub 2014 Feb 14.
  • the method for separating rAAVhu68 (or AAVrh91 or AAVhu95) particles having packaged genomic sequences from genome-deficient AAVhu68 (or AAVrh91 or AAVhu95) intermediates involves subjecting a suspension comprising recombinant AAVhu68 (or rh91) viral particles and AAVhu68 (or AAVrh91 or AVhu95) capsid intermediates to fast performance liquid chromatography, wherein the AAVhu68 (or AAVrh91 or AAVhu95) viral particles and AAVhu68 intermediates are bound to a strong anion exchange resin equilibrated at a pH of about 10.2 (or about 9.8 for AAVrh91), and subjected to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 nanometers (nm) and about 280 nm.
  • the pH may be in the range of about 10 to 10.4.
  • the AAV full capsids are collected from a fraction which is eluted when the ratio of A260/A280 reaches an inflection point.
  • the diafiltered product may be applied to an affinity resin (Life Technologies) that efficiently captures the AAV serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while AAV particles are efficiently captured.
  • the rAAV.hCDKL5 (e.g., rAAV.BIP-TATk28.hCDKL5) is suspended in a suitable physiologically compatible composition (e.g., a buffered saline).
  • a suitable physiologically compatible composition e.g., a buffered saline
  • This composition may be frozen for storage, later thawed and optionally diluted with a suitable diluent.
  • the vector may be prepared as a composition which is suitable for delivery to a patient without proceeding through the freezing and thawing steps.
  • NAb titer a measurement of how much neutralizing antibody (e.g., anti-AAV Nab) is produced which neutralizes the physiologic effect of its targeted epitope (e.g., an AAV).
  • Anti-AAV NAb titers may be measured as described in, e.g., Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno- Associated Viruses. Journal of Infectious Diseases, 2009. 199(3): p. 381-390, which is incorporated by reference herein.
  • sc refers to self-complementary.
  • Self-complementary AAV refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template.
  • dsDNA double stranded DNA
  • a “replication-defective virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells.
  • the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the gene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication.
  • rAAV particles are referred to as DNase resistant.
  • DNase endonuclease
  • other endo- and exo- nucleases may also be used in the purification steps described herein, to remove contaminating nucleic acids.
  • Such nucleases may be selected to degrade single stranded DNA and/or double-stranded DNA, and RNA.
  • Such steps may contain a single nuclease, or mixtures of nucleases directed to different targets, and may be endonucleases or exonucleases.
  • nuclease-resistant indicates that the AAV capsid has fully assembled around the expression cassette which is designed to deliver a gene to a host cell and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process.
  • the vector is a viral vector selected from a recombinant parvovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adenovirus; or a non-viral vector selected from naked DNA, naked RNA, an inorganic particle, a lipid particle, a polymer-based vector, or a chitosan-based formulation.
  • the selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • a “replication-defective virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells.
  • the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the transgene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production.
  • replication-defective viruses may be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrating or non-integrating), or another suitable virus source.
  • AAV adeno-associated viruses
  • adenoviruses adenoviruses
  • lentiviruses integrating or non-integrating
  • a “nucleic acid”, as described herein, can be RNA, DNA, or a modification thereof, and can be single or double stranded, and can be selected, for example, from a group including: nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide-nucleic acid (PNA), pseudocomplementary PNA (pc-PNA), locked nucleic acid (LNA) etc.
  • PNA peptide-nucleic acid
  • pc-PNA pseudocomplementary PNA
  • LNA locked nucleic acid
  • nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi ), antisense oligonucleotides etc.
  • sequence identity refers to the residues in the two sequences which are the same when aligned for correspondence.
  • the length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g., of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.
  • Percent identity may be readily determined for amino acid sequences over the full- length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequences.
  • a suitable amino acid fragment may be at least about 8 amino acids in length, and may be up to about 700 amino acids.
  • identity when referring to “identity”, “homology”, or “similarity” between two different sequences, identity , homology or similarity is determined in reference to “aligned” sequences. “Aligned” sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.
  • Sequence alignment programs are available for amino acid sequences, e.g., the “Clustal X”, “Clustal Omega” “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13): 2682-2690 (1999).
  • nucleic acid sequences are also available for nucleic acid sequences. Examples of such programs include, “Clustal W”, “Clustal Omega”, “CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using FastaTM, a program in GCG Version 6.1. FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using FastaTM with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
  • FastaTM provides alignments and percent sequence identity of the regions
  • compositions Provided herein is a composition comprising an rAAV or a vector as described herein and an aqueous suspension media.
  • the suspension is formulated for intravenous delivery, intrathecal administration, or intracerebroventricular administration.
  • compositions containing at least one rAAV stock and an optional carrier, excipient and/or preservative are provided herein.
  • a “stock” of rAAV refers to a population of rAAV. Despite heterogeneity in their capsid proteins due to deamidation, rAAV in a stock are expected to share an identical vector genome.
  • a stock can include rAAV having capsids with, for example, heterogeneous deamidation patterns characteristic of the selected AAV capsid proteins and a selected production system. The stock may be produced from a single production system or pooled from multiple runs of the production system. A variety of production systems, including but not limited to those described herein, may be selected.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary' active ingredients can also be incorporated into the compositions.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells.
  • the rAAV vector delivered vector genomes may be formulated for delivery' either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or tire like.
  • a composition in one embodiment, includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.
  • a surfactant are present in the formulation.
  • the composition may be transported as a concentrate which is diluted for administration to a subject.
  • the composition may be lyophilized and reconstituted at the time of administration.
  • a suitable surfactant, or combination of surfactants may be selected from among non-ionic surfactants that are nontoxic.
  • a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH, has an average molecular weight of 8400.
  • Pluronic® F68 [BASF] also known as Poloxamer 188
  • Other surfactants and other Poloxamers may be selected, i.e.
  • nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (polyethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Poly oxy capryllic glyceride), poly oxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol.
  • the formulation contains a poloxamer.
  • Poloxamer 188 is selected.
  • the surfactant may be present in an amount up to about 0.0005 % to about 0.001% (based on weight ratio, w/w %) of the suspension. In another embodiment, the surfactant may be present in an amount up to about 0.0005 % to about 0.001% (based on volume ratio, v/v %) of the suspension. In yet another embodiment, the surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension, wherein n % indicates n gram per 100 mL of the suspension.
  • the composition includes a carrier, diluent, excipient and/or adjuvant.
  • Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.
  • the buffer/carrier should include a component that prevents the rAAV, from sticking to the infusion tubing but does not interfere with the rAAV binding activity in vivo.
  • a suitable surfactant, or combination of surfactants may be selected from among non-ionic surfactants that are nontoxic.
  • a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Poloxamer 188 (also known under the commercial names Pluronic® F68 [BASF], Lutrol® F68, Synperonic® F68, Kolliphor® P188) which has a neutral pH, has an average molecular weight of 8400.
  • Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (polyethylene oxide)), SOLUTOL HS 15 (Macrogol- 15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), polyoxy -oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol.
  • the formulation contains a poloxamer.
  • copolymers are commonly named with the letter "P" (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the poly oxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content.
  • Poloxamer 188 is selected.
  • the surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.
  • the composition containing the rAAV.hCDKL5 is delivered at a pH in the range of 6.8 to 8, or 7.2 to 7.8, or 7.5 to 8.
  • a pH above 7.5 may be desired, e.g., 7.5 to 8, or 7.8.
  • the formulation may contain a buffered saline aqueous solution not comprising sodium bicarbonate.
  • a buffered saline aqueous solution comprising one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and mixtures thereof, in water, such as a Harvard’s buffer.
  • the aqueous solution may further contain Kolliphor® Pl 88, a poloxamer which is commercially available from BASF which was formerly sold under the trade name Lutrol® F68.
  • the aqueous solution may have a pH of 7.2.
  • the formulation may contain a buffered saline aqueous solution comprising 1 mM Sodium Phosphate (NasPOr), 150 mM sodium chloride (NaCl), 3mM potassium chloride (KC1), 1.4 mM calcium chloride (CaCh), 0.8 mM magnesium chloride (MgCh), and 0.001% poloxamer (e.g., Kolliphor®) 188, pH 7.2. See, e.g., harvardapparatus.com/harvard-apparatus-perfusion-fluid.html.
  • Harvard’s buffer is preferred due to better pH stability observed with Harvard’s buffer.
  • the formulation buffer is artificial CSF with Pluronic F68.
  • the formulation may contain one or more permeation enhancers.
  • suitable permeation enhancers may include, e.g., mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-laurel ether, or EDTA.
  • compositions of the invention may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
  • suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • compositions according to the present invention may comprise a pharmaceutically acceptable carrier, such as defined above.
  • the compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and/or admixed with suitable excipients designed for delivery to the subject via injection, osmotic pump, intrathecal catheter, or for delivery by another device or route.
  • the om maya reservoir is used for delivery.
  • the composition is formulated for intrathecal delivery.
  • the composition is formulated for intravenous (iv) delivery.
  • a method of treating CDD comprising administrating an effective amount of an rAAV or a vector as described herein to a subject in need thereof.
  • the vectors provided herein encoding BIP-TATk28-hCDKL5 may be used in a regimen involving co-administration with the BIP-TATk28- hCDKL5 protein and/or combined with other CDKL5 therapies.
  • an “effective amount” herein is the amount which achieves amelioration of CDD symptoms and/or delayed CDD progression.
  • the vectors are administered in sufficient amounts to transfect the cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue adverse effects, or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts.
  • Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a desired organ (e.g., brain, CSF, the liver (optionally via the hepatic artery), lung, heart, eye, kidney,), oral, inhalation, intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, intraparenchymal, intracerebroventricular, intrathecal, ICM, lumbar puncture and other parenteral routes of administration. Routes of administration may be combined, if desired.
  • Dosages of the viral vector depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and can thus vary among patients.
  • a therapeutically effective human dosage of the viral vector is generally in the range of from about 25 to about 1000 microliters to about 100 mL of solution containing concentrations of from about 1 x 10 9 to 1 x 10 16 vector genome copies.
  • a volume of about 1 mL to about 15 mL, or about 2.5 mL to about 10 mL, or about 5 mL suspension is delivered.
  • a volume of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 mL suspension is delivered.
  • a dose of about 8.9 x 10 12 to 2.7 x 10 14 GC total is administered in this volume.
  • a dose of about 1.1 xlO 10 GC/g brain mass to about 3.3 x 10 11 GC/g brain mass is administered in this volume.
  • the dosage is adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed.
  • the levels of expression of the transgene product can be monitored to determine the frequency of dosage resulting in viral vectors, preferably AAV vectors containing the minigene.
  • dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using the compositions of the invention.
  • the replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 10 9 GC to about 1.0 x 10 16 GC (to treat an subject) including all integers or fractional amounts within the range, and preferably 1.0 x 10 12 GC to 1.0 x 10 14 GC for a human patient.
  • the compositions are formulated to contain at least IxlO 9 , 2xl0 9 , 3xl0 9 , 4xl0 9 , 5xl0 9 , 6xl0 9 , 7xl0 9 , 8xl0 9 , or 9xlO 9 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least IxlO 10 , 2xlO 10 , 3xl0 10 , 4xlO 10 , 5xl0 10 , 6xlO 10 , 7xlO 10 , 8xl0 10 , or 9xlO 10 GC per dose including all integers or fractional amounts within the range.
  • the compositions are formulated to contain at least IxlO 11 , 2xlO n , 3xl0 n , 4xlO n , 5xl0 n , 6xlO n , 7xlO n , 8xl0 n , or 9xlO n GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least IxlO 12 , 2xl0 12 , 3xl0 12 , 4xl0 12 , 5xl0 12 , 6xl0 12 , 7xl0 12 , 8xl0 12 , or 9xl0 12 GC per dose including all integers or fractional amounts within the range.
  • the compositions are formulated to contain at least IxlO 13 , 2xl0 13 , 3xl0 13 , 4xl0 13 , 5xl0 13 , 6xl0 13 , 7xl0 13 , 8xl0 13 , or 9xl0 13 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least IxlO 14 , 2xl0 14 , 3xl0 14 , 4xl0 14 , 5xl0 14 , 6xl0 14 , 7xl0 14 , 8xl0 14 , or 9xl0 14 GC per dose including all integers or fractional amounts within the range.
  • the compositions are formulated to contain at least IxlO 15 , 2xl0 15 , 3xl0 15 , 4xl0 15 , 5xl0 15 , 6xl0 15 , 7xl0 15 , 8xl0 15 , or 9xl0 15 GC per dose including all integers or fractional amounts within the range.
  • the dose can range from IxlO 10 to about IxlO 15 GC per kg body weight including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 9 , 2xl0 9 , 3xl0 9 , 4xl0 9 , 5xl0 9 , 6xl0 9 , 7xl0 9 , 8xl0 9 , or 9xl0 9 GC per kg body weight including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 10 , 2xlO 10 , 3xl0 10 , 4xlO 10 , 5xl0 10 , 6xlO 10 , 7xlO 10 , 8xl0 10 , or 9xlO 10 GC per kg body weight including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 11 , 2xlO n , 3xl0 n , 4xlO n , 5xl0 n , 6xlO n , 7xlO n , 8xl0 n , or 9xlO n GC per kg body weight including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 12 , 2xl0 12 , 3xl0 12 , 4xl0 12 , 5xl0 12 , 6xl0 12 , 7xl0 12 , 8xl0 12 , or 9xl0 12 GC per kg body weight including all integers or fractional amounts within the range.
  • the effective amount of the vector is about 1x10 , 2x10 , 3x10 , 4x10 , 5xl0 13 , 6xl0 13 , 7xl0 13 , 8xl0 13 , or 9xl0 13 GC per kg body weight including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 14 , 2xl0 14 , 3xl0 14 , 4xl0 14 , 5xl0 14 , 6xl0 14 , 7xl0 14 , 8xl0 14 , or 9xl0 14 GC per kg body weight including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 15 , 2xl0 15 , 3xl0 15 , 4xl0 15 , 5xl0 15 , 6xl0 15 , 7xl0 15 , 8xl0 15 , or 9xl0 15 GC per kg body weight including all integers or fractional amounts within the range.
  • the dose can range from IxlO 10 to about IxlO 15 GC per gram (g) brain mass including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 9 , 2xl0 9 , 3xl0 9 , 4xl0 9 , 5xl0 9 , 6xl0 9 , 7xl0 9 , 8xl0 9 , or 9xl0 9 GC per gram (g) brain mass including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 10 , 2xlO 10 , 3xl0 10 , 4xlO 10 , 5xl0 10 , 6xlO 10 , 7xlO 10 , 8xl0 10 , or 9x10 10 GC per gram (g) brain mass including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 11 , 2xlO n , 3x10”, 4x10”, 5x10”, 6x10”, 7xlO n , 8xl0 n , or 9x10“ GC per gram (g) brain mass including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 12 , 2xl0 12 , 3xl0 12 , 4xl0 12 , 5xl0 12 , 6xl0 12 , 7xl0 12 , 8xl0 12 , or 9xl0 12 GC per gram (g) brain mass including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 13 , 2xl0 13 , 3xl0 13 , 4xl0 13 , 5xl0 13 , 6xl0 13 , 7xl0 13 , 8xl0 13 , or 9xl0 13 GC per gram (g) brain mass including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 14 , 2xl0 14 , 3xl0 14 , 4xl0 14 , 5xl0 14 , 6xl0 14 , 7xl0 14 , 8xl0 14 , or 9xl0 14 GC per gram (g) brain mass including all integers or fractional amounts within the range.
  • the effective amount of the vector is about IxlO 15 , 2xl0 15 , 3xl0 15 , 4xl0 15 , 5xl0 15 , 6xl0 15 , 7xl0 15 , 8xl0 15 , or 9xl0 15 GC per gram (g) brain mass including all integers or fractional amounts within the range.
  • the volume of earner, excipient or buffer is at least about 25 pL. In one embodiment, the volume is about 50 pL. In another embodiment, the volume is about 75 pL. In another embodiment, the volume is about 100 pL. In another embodiment, the volume is about 125 pL. In another embodiment, the volume is about 150 pL. In another embodiment, the volume is about 175 pL.
  • the volume is about 200 pL. In another embodiment, the volume is about 225 pL. In yet another embodiment, the volume is about 250 pL. In yet another embodiment, the volume is about 275 pL. In yet another embodiment, the volume is about 300 pL. In yet another embodiment, the volume is about 325 pL. In another embodiment, the volume is about 350 pL. In another embodiment, the volume is about 375 pL. In another embodiment, the volume is about 400 pL. In another embodiment, the volume is about 450 pL. In another embodiment, the volume is about 500 pL. In another embodiment, the volume is about 550 pL. In another embodiment, the volume is about 600 pL. In another embodiment, the volume is about 650 pL. In another embodiment, the volume is about 700 pL. In another embodiment, the volume is between about 700 and 1000 pL.
  • the dose may be in the range of about 1 x 10 9 GC/g brain mass to about 1 x 10 12 GC/g brain mass. In certain embodiments, the dose may be in the range of about 1 x 10 10 GC/g brain mass to about 3 x 10 11 GC/g brain mass. In certain embodiments, the dose may be in the range of about 1 x 10 10 GC/g brain mass to about 2.5 x 10 11 GC/g brain mass. In certain embodiments, the dose may be in the range of about 5 x 10 10 GC/g brain mass.
  • the viral constructs may be delivered in doses of from at least about least IxlO 9 GC to about 1 x 10 15 , or about 1 x 10 11 to 5 x 10 13 GC.
  • Suitable volumes for delivery of these doses and concentrations may be determined by one of skill in the art. For example, volumes of about 1 pL to 150 mL may be selected, with the higher volumes being selected for adults. Typically, for newborn infants a suitable volume is about 0.5 mL to about 10 mL, for older infants, about 0.5 mL to about 15 mL may be selected. For toddlers, a volume of about 0.5 mL to about 20 mL may be selected. For children, volumes of up to about 30 mL may be selected.
  • volume up to about 50 mL may be selected.
  • a patient may receive an intrathecal administration in a volume of about 5 mL to about 15 mL are selected, or about 7.5 mL to about 10 mL.
  • Other suitable volumes and dosages may be determined. The dosage may be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed.
  • the above-described recombinant vectors may be delivered to host cells according to published methods.
  • the rAAV preferably suspended in a physiologically compatible carrier, may be administered to a human or non-human mammalian patient.
  • the rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or mixture of salts.
  • the formulation is adjusted to a physiologically acceptable pH, e.g., in the range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8.
  • pH of the cerebrospinal fluid is about 7.28 to about 7.32
  • a pH within this range may be desired; whereas for intravenous delivery, a pH of about 6.8 to about 7.2 may be desired.
  • other pHs within the broadest ranges and these subranges may be selected for other route of delivery.
  • Intrathecal delivery refers to a route of administration for drugs via an injection 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/intracistemal, and/or Cl-2 puncture.
  • material may be introduced for diffusion throughout the subarachnoid space by means of lumbar puncture.
  • injection may be into the cistema magna.
  • a rAAV, vector, or composition as described herein is administrated to a subject in need via the intrathecal administration.
  • the intrathecal administration is performed as described in US Patent Publication No. 2018-0339065 Al, published November 29, 2019, which is incorporated herein by reference in its entirety.
  • intracistemal delivery or “intracisternal administration” refer to a route of administration for drugs directly into the cerebrospinal fluid of the cistema magna cerebellomedularis, more specifically via a suboccipital puncture or by direct injection into the cistema magna or via permanently positioned tube.
  • treatment of the composition described herein has minimal to mild asymptomatic degeneration of DRG sensory neurons in animals and/or in human patients, well-tolerated with respect to sensory nerve toxicity and subclinical sensory neuron lesions.
  • the vectors provided herein may be administered intrathecally via the method and/or the device provided in this section and described in WO 2018/160582, which is incorporated by reference herein. Alternatively, other devices and methods may be selected.
  • the method comprises the steps of CT-guided suboccipital injection via spinal needle into the cisterna magna of a patient.
  • CT Computed Tomography
  • the term Computed Tomography (CT) refers to radiography in which a three-dimensional image of a body structure is constructed by computer from a series of plane cross-sectional images made along an axis.
  • vectors and/or compositions thereof as described herein are administered via computed tomography- (CT-) guided sub-occipital injection into the cistema magna (intra-cistema magna [ICM]).
  • CT- computed tomography-
  • ICM intra-cistema magna
  • the Ommaya Reservoir is used for delivery of a pharmaceutical composition.
  • the apparatus is described in US Patent Publication No. 2018-0339065 Al, published November 29, 2019, which is incorporated herein by reference in its entirety.
  • “Patient” or “subject”, as used herein interchangeably, means a male or female mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research.
  • the subject of these methods and compositions is a human patient.
  • the subject of these methods and compositions is a male or female human.
  • the subject of these methods and compositions is diagnosed with CDD and/or with symptoms of CDD.
  • the methods and compositions may be used for treatment of any of the stages of CDD.
  • the patient is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 month(s) old, or about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18 year(s) old.
  • the patient is a toddler, e.g., 18 months to 3 years of age.
  • the patient is from 3 years to 6 years of age, from 3 years to 12 years of age, from 3 years to 18 years of age, from 3 years to 30 years of age.
  • patients are older than 18 years of age.
  • Symptoms in CDD include seizures that usually begin within the first 3 months of life, and can appear as early as the first week after birth.
  • the types of seizures change with age, and may follow a predictable pattern.
  • the most common types are generalized tonic- clonic seizures, which involve a loss of consciousness, muscle rigidity, and convulsions; tonic seizures, which are characterized by abnormal muscle contractions; and epileptic spasms, which involve short episodes of muscle jerks.
  • Seizures occur daily in most people with CDKL5 deficiency disorder, although they can have periods when they are seizure-free. Seizures in CDKL5 deficiency disorder are typically resistant to treatment.
  • CDKL5 deficiency disorder Other common features include repetitive hand movements (stereotypies), such as clapping, hand licking, and hand sucking; teeth grinding (bruxism); disrupted sleep; feeding difficulties; and gastrointestinal problems including constipation and backflow of acidic stomach contents into the esophagus (gastroesophageal reflux). Some affected individuals have episodes of irregular breathing. Distinctive facial features in some people with CDKL5 deficiency disorder include a high and broad forehead, large and deep-set eyes, a well-defined space between the nose and upper lip (philtrum), full lips, widely spaced teeth, and a high roof of the mouth (palate). Other physical differences can also occur, such as an unusually small head size (microcephaly), side-to-side curvature of the spine (scoliosis), and tapered fingers.
  • the terms “increase” “decrease” “reduce” “ameliorate” “improve” “delay” or any grammatical variation thereof, or any similar terms indication a change means a variation of about 5 fold, about 2 fold, about 1 fold, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5 % compared to the corresponding reference (e.g., untreated control or a subject in normal condition without CDD), unless otherwise specified.
  • the patient receives medications controlling some signs and symptoms associated with the CDD, such as seizures, muscle stiffness, or problems with breathing, sleep, the gastrointestinal tract or the heart.
  • the seizures are tonic-clonic seizures, which involve a loss of consciousness, muscle rigidity, and convulsions or tonic seizures, which are characterized by abnormal muscle contractions.
  • symptoms include epileptic spasms, which involve short episodes of muscle jerks.
  • Other symptoms include repetitive hand movements, such as clapping, hand licking, and hand sucking; teeth grinding; disrupted sleep; feeding difficulties; gastrointestinal problems including constipation and backflow of acidic stomach contents into the esophagus (gastroesophageal reflux); and/or irregular breathing.
  • a diuretic agent may be used in co-therapy in a subject in need thereof.
  • Diuretic agent used may be acetazolamine (Diamox) or other suitable diuretics.
  • the diuretic agent is administered at the time of gene therapy administration. In some embodiments, the diuretic agent is administered prior to gene therapy administration. In some, embodiments the diuretic agent is administered where the volume of injection is 3 mL.
  • co-therapies may be utilized, which comprise coadministration of Cdkl5-isoform 1, isoform 2, isoform 3, and/or isoform 4- expressing vectors, or various two- or three-way combinations thereof.
  • co-therapy may further comprise administration of another active agent.
  • co-therapy may comprise enzyme replacement therapy.
  • an immunosuppressive co-therapy may be used in a subject in need.
  • Immunosuppressants for such co-therapy include, but are not limited to, a glucocorticoid, steroids, antimetabolites, T-cell inhibitors, a macrolide (e.g., a rapamycin or rapalog), and cytostatic agents including an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, an antibody, or an agent active on immunophilin.
  • the immune suppressant may include a nitrogen mustard, nitrosourea, platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD3-directed antibodies, anti-IL-2 antibodies, ciclosporin, tacrolimus, sirolimus, IFN- , IFN-y, an opioid, or TNF-a (tumor necrosis factor- alpha) binding agent.
  • the immunosuppressive therapy may be started 0, 1, 2, 3, 4, 5, 6, 7, or more days prior to or after the gene therapy administration.
  • Such immunosuppressive therapy may involve administration of one, two or more drugs (e.g., glucocorticoids, prednelisone, micophenolate mofetil (MMF) and/or sirolimus (i.e., rapamycin)).
  • drugs e.g., glucocorticoids, prednelisone, micophenolate mofetil (MMF) and/or sirolimus (i.e., rapamycin
  • Such immunosuppressive drugs may be administrated to a subject in need once, twice or for more times at the same dose or an adjusted dose.
  • Such therapy may involve co-administration of two or more drugs, the (e.g., prednelisone, micophenolate mofetil (MMF) and/or sirolimus (i.e. , rapamycin)) on the same day.
  • One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose.
  • Such therapy may be for about 1
  • RNA Ribonucleic acid
  • expression is used herein in its broadest meaning and comprises the production of RNA or of RNA and protein.
  • expression or “translation” relates in particular to the production of peptides or proteins. Expression may be transient or may be stable.
  • a refers to one or more, for example, “an enhancer”, is understood to represent one or more enhancer(s).
  • the terms “a” (or “an”), “one or more,” and “at least one” is used interchangeably herein.
  • the term “about” when used to modify a numerical value means a variation of ⁇ 10%, ( ⁇ 10%, e.g., ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8, ⁇ 9, ⁇ 10, or values therebetween) from the reference given, unless otherwise specified.
  • technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.
  • CDD Several models for CDD have been developed and may selected for use in evaluating therapeutic effect. These following models are null for CDKL5 expression: g., a Cdkl5-ko mouse having a deletion in exon 6 (A exon 6), Wang et al, Proceedings of the National Academy of Sciences Dec 2012, 109 (52) 21516-21521; DOI: 10. 1073/pnas. 1216988110; a Cdkl5-ko mouse having a deletion in exon 4 (A exon 4) (see, Amendola et al. (2014) Mapping Pathological Phenotypes in a Mouse Model of CDKL5 Disorder. PLoS ONE 9(5): e91613.
  • a fusion protein comprising an exogenous leader and cell penetrating peptide fused to engineered sequences encoding human CDKLK5 isoform 1 were generated and comparative studies were performed with the corresponding construct without the exogenous BIP-TAT sequences, containing the native leader sequence.
  • the rAAV are generated using triple transfection techniques, utilizing (1) a cis plasmid encoding AAV2 rep proteins and the AAVhu68 VP1 cap gene, (2) a cis plasmid comprising adenovirus helper genes not provided by the packaging cell line which expresses adenovirus El a, and (3) a trans plasmid containing the vector genome for packaging in the AAV capsid.
  • the trans plasmid is designed to contain either the vector genome of BIP-TAT28k28-CDKL5 as illustrated in FIG 1 or the comparative vector genome containing the sequences encoding hCDKL5 with the native hCDKL5 leader peptide.
  • FIG. 1 shows a graphic map of the vector genome in a recombinant AAV particle.
  • the vector genome contains an AAV 5’ inverted terminal repeat (ITR) and an AAV 3’ ITR at the extreme 5’ and 3’ end, respectively.
  • the ITRs flank the sequences of the expression cassette packaged into the AAV capsid which have sequences encoding a BIP-TATk28-hCDKL5 fusion protein, in which BIP refers to a signal peptide and TATk28 (also referenced to as TAT) is a cell penetrating peptide.
  • the expression cassette further comprises regulatory sequences operably linked to the fusion protein coding sequences, including a hybrid chicken beta actin (CBH) promoter, and an SV40 poly A.
  • CBH hybrid chicken beta actin
  • FIG 2 provides an illustration of cross-correction, in which a secretable BIP-TAT-hCDKL5 fusion protein is delivered via an AAV which corrects deficient hCDKL5 in vector- transduced cells and delivers a secreted hCDKL5 protein to other brain cells.
  • AAVhu68.BiP. TATk28.CDKL5 achieved broader brain CDKL5 delivery and expression for CDKL5-deficiency disorder (CDD).
  • mice were administered with AAVhu68.CBH.BIP-TAT28k- hCDKL5co2.SV40 or AAVhu68.CBH.hCDKL5co2.SV40 (control) at a dose of 1 x 10 11 (lei 1) GC/mouse via intraventricular (ICV) injection, at age P0 or P14.
  • ICV intraventricular
  • mice were necropsied and tissues were collected.
  • Dual immunofluorescence (IF) and in-situ hybridization (ISH) were performed for expression and quantification studies.
  • IF immunofluorescence
  • ISH in-situ hybridization
  • FIG. 3A shows western blot analysis of BIP-TATk28-hCDKL5 protein expression in brain of male mice of ID Nos: 734 and 738 (WT PBS), 753 (CDKL5 /y PBS), 783, 855 and 860 (CDKL5 /y BIP.TATk28.hCDKL5), 772 and 832 (CDKL5 /y CDKL5).
  • 3B shows western blot analysis of BIP-TATk28-hCDKL5 protein expression in brain of male mice of ID Nos: 1157 and 1162 (WT PBS), 1156 and 1160 (CDKL5 /y PBS), 980 and 1136 (CDKL5 /y BIP.TATk28.hCDKL5), 1202 and 1206 (CDKL5 /y CDKL5).
  • WT PBS 1157 and 1162
  • 1156 and 1160 CDKL5 /y PBS
  • 980 and 1136 CDKL5 /y BIP.TATk28.hCDKL5
  • 1202 and 1206 CDKL5 /y CDKL5
  • Male neonatal control or CDKL5 /y mice were injected ICV with PBS or AAVhu68-CBH-BIP-TATk28-hCDKL5 vector or AAVhu68-CBH-hCDKL5 vector.
  • Vector dose was 1 x 10 11 (
  • FIG. 3A shows a loading control below a CDKL5 immunoblot.
  • FIG. 3C shows a graph with densitometric quantitation of CDKL5 expression.
  • BIP-TATk28-hCDKL5 protein expression reached 50 percent of wild type (WT) PBS controls and was higher than untagged CDKL5 expression.
  • FIG. 3D shows BIP-TATk28-CDKL5co2 and hCDKL5co2 mRNA expression levels plotted as hCDKL5-co2 transcript per lOOng mRNA.
  • FIG. 17A further shows cortical protein expression (CDKL5) in mice, plotted as percent wild-type-PBS expression in CDKL5 /y with P14 treatment.
  • FIG. 17B further shows cortical mRNA expression, more specifically, BIP- TATk28-CDKL5 (BTC) and hCDKL5 mRNA expression levels plotted as hCDKL5-co2 transcript per lOOng mRNA.
  • mice Male neonatal control or CDKL5 /y mice were injected ICV with PBS or AAVhu68-CBH-BIP-TATk28-hCDKL5 vector or AAVhu68-CBH-hCDKL5 vector.
  • Vector dose was lel l (IxlO 11 ) genome copies per animal. After 1 month in life brains were harvested and subject to dual immunofluorescence and in situ hybridization analysis.
  • FIG. 4 shows a representative fluorescent images of sagittal sections of cortex from AAVhu68- CBH-BIP-TATk28-hCDKL5co2 treated CDKL5 /y mice showing the distribution of BIP- TATk28-hCDKL5 protein (yellow) and BIP-TATk28-hCDKL5 mRNA (blue). Nuclei (purple) are counterstained with DAPI (4',6-diamidino-2-phenylindole).
  • FIG. 18 further shows representative fluorescent images (Immunofluorescence and In Situ Hybridization) of cortex from AAVhu68-CBH-BIP-TATk28-hCDKL5co2 treated CDKL5 /y mice showing the distribution of BIP-TATk28-hCDKL5 protein (yellow) and BIP-TATk28-hCDKL5 mRNA (blue). Nuclei (purple) are counterstained with DAPI (4',6-diamidino-2-phenylindole). Arrows highlight cross corrected cells which are positive for CDKL5 protein only.
  • CDKL5 protein was identified by immunofluorescence while BIP-TATk28-hCDKL5 mRNA was identified by in situ hybridization using a custom RNAscope probe. These results show the increased CDKL5 expression and cross-correction in the cortex of CDKL5 /y mice treated at P14 with BIP-TATk28-hCDKL5.
  • CDKL5 is made within the cell following expression from the rAAV-CBH-BIP-TATk28-hCDKL5 vector, wherein a subset of the expressed BIP-TATk28-hCDKL5 is secreted and taken up by neighboring cells. This approach allows for cross correction for many surrounding cells in addition to the cells into which hCDKL5 gene was transduced.
  • FIG. 5A shows the number of cross corrected cells positive for Cdkl5 protein that were quantitated using Visiopharm® software.
  • FIG. 5C further shows the number of cross corrected cells positive for Cdkl5 protein in neonatal and CDKL5-/y injected mice that were quantitated using Visiopharm® software, more specifically in multiple brain regions of P14 BIP-TATk28-hCDKL5 treated CDKL5-/y mice.
  • FIG. 5B further displays the number of cells positive for Cdkl5 protein (with or without a signal for Cdkl5 mRNA) in the cortex, striatum, thalamus, hippocampus and hypothalamus quantitated using Visiopharm® pathology image analysis software.
  • the mean fraction of cross corrected cells in the cortex was 2.46 %.
  • the mean fraction of cross corrected cells in the striatum was 2.71 %.
  • the mean fraction of cross corrected cells in the thalamus was 1.37 %.
  • the mean fraction of cross corrected cells in the hippocampus was 4.8 %.
  • the mean fraction of cross corrected cells in the hypothalamus was 0.24 %.
  • FIG. 19A shows a representative microscopy image of CDKL5 expression in hippocampal cross-section tissue of female control CDKL5 +/_ mice administered with PBS.
  • FIG. 19B shows a representative microscopy image of CDKL5 expression in hippocampal cross-section tissue of female control CDKL5 +/_ mice treated with AAV.BIP-TATk28-hCDKL5.
  • FIG. 19C shows hippocampal BIP-TATk28- hCDKL5 expression, plotted as percent wildtype-PBS expression.
  • FIG. 19D shows western blot analysis of BIP-TATk28-hCDKL5 and endogenous CDKL5 protein expression (in comparison with the total protein control) in hippocampal tissue of female CDKL5 +/_ mice.
  • mice brain CDKL5 protein levels rise around two weeks of age; therefore, to mimic this natural increase we retroorbitally injected P18 CDKL5 /y mice with PHP.B-CBH-BIP-TATk28-CDKL5 at a dose of lei 1 (IxlO 11 ) GC/animal.
  • mice reached 3 months of age we tested the impact of treatment on cage locomotor activity using an open field testing system (FIG 7) and harvested brains for determination of BIP-TATk28-CDKL5 protein expression by Western immunoblot (FIG 6).
  • BIP-TATk28-CDKL5 protein was strongly expressed in CDKL5 /y mice with little expression in treated control mice (FIG 6).
  • EEG electroencephalography
  • FIG 11 A shows that from 12 pm to 1 pm during the light cycle, the relative power of delta frequencies (0.5-4 Hz) was significantly reduced in young female CDKL5 +/_ mice compared to wild type (WT) controls.
  • CDKL5 +/_ mice also showed both significant reductions and increases in theta (4-8 Hz) frequency power and an increase in alpha (8-13 Hz) frequency power.
  • Beta frequency power (13-24 Hz) was unaffected.
  • FIG 1 IB shows that from 12 pm to 1 pm during the light cycle, the relative power of gamma frequencies (25-50 Hz) was modestly but significantly increased in young female CDKL5 +/_ mice compared with wild type (WT) controls.
  • FIG 12A shows that from 12 am to 1 am during the dark cycle, delta frequency (0.5- 4 Hz) power was significantly reduced in young female CDKL5 +/_ mice compared to wild type (WT) controls.
  • CDKL5 +/_ mice also showed both significant reductions and increases in theta (4-8 Hz) frequency power and an increase in alpha (8-13 Hz) frequency power.
  • Beta frequency power 13-24 Hz was unaffected.
  • FIG 12B shows that from 12 am to lam during the dark cycle, the relative power of gamma frequencies (25-50 Hz) was modestly but significantly increased in young female CDKL5 +/_ mice compared with wild type (WT) controls.
  • FIG. 13Aand 13B show that BIP-TATk28k-hCDKL5 reduces aberrant delta/theta and gamma power in CDKL5 +/_ mice during the middle of the light cycle (12pm - 2pm window).
  • FIG. 13A shows the fraction of total power output at delta (0-4 Hz), theta (4-8 Hz), alpha (8-13 Hz) and beta (13-24 Hz) frequency bands. The fraction of delta, theta power was decreased in PBS treated CDKL5 +/_ mice compared with PBS treated wild type controls. The fraction of alpha power in CDKL5 +/_ mice was increased relative to wild type controls. BIP- TATk28-hCDKL5 vector treatment significantly increased delta and theta power towards control levels.
  • FIG. 13B shows the fraction of total pro-seizure gamma (15-50 Hz) power output.
  • Gamma power was significantly elevated in PBS treated CDKL5 +/_ mice compared with PBS treated wild type controls.
  • BIP-TATk28- hCDKL5 vector treatment significantly decreased gamma power in CDKL5 +/_ mice but not WT control mice.
  • FIG. 14A and 14B show that BIP-TATk28k-hCDKL5 reduces abnormalities in delta and gamma frequency bands in CDKL5 +/_ mice at the dark to light transition (6am-7am window). EEG activity was analyzed between 6 am and 7 am when mice are transitioning to low activity and sleep states.
  • FIG. 14A shows the fraction of total power output at delta (0-4 Hz), theta (4-8 Hz), alpha (8-13 Hz) and beta (13-24 Hz) frequency bands. The fraction of delta, theta power was decreased in PBS treated CDKL5 +/_ mice compared with PBS treated wild type controls. The fraction of alpha power in CDKL5 +/_ mice was increased relative to wild type controls.
  • FIG. 14B shows the fraction of total pro-seizure gamma (15-50 Hz) power output. Heightened gamma frequency band activity occurs in Cdkl5 -deficient patients (Ren et al., 2015) and in Cdkl5 deficient mice (Mulcahey et al., 2020). Gamma power was significantly elevated in PBS treated CDKL5 +/_ mice compared with PBS treated wild type controls. BIP-TATk28-hCDKL5 vector treatment significantly decreased gamma power in CDKL5 +/_ mice but not WT control mice.
  • FIG. 15A and 15B shows that BIP-TATk28k-hCDKL5 reduces abnormalities in theta, alpha, and gamma frequencies in CDKL5 +/_ mice during the light to dark transition (6pm-7pm window).
  • FIG. 15A shows the fraction of total power output at delta (0-4 Hz), theta (4-8 Hz), alpha (8-13 Hz) and beta (13-24 Hz) frequency bands. The fraction of delta, low theta power was decreased in PBS treated CDKL5 +/_ mice compared with PBS treated wild type controls. The fraction of alpha power and high theta power in CDKL5 +/_ mice were increased relative to wild type controls.
  • FIG. 15B shows the fraction of gamma (15-50 Hz) power output.
  • Gamma power was significantly elevated in PBS treated CDKL5 +/_ mice compared with PBS treated wild type controls.
  • BIP-TATk28-hCDKL5 vector treatment significantly decreased gamma power, particularly in the 31-38 Hz range, in CDKL5 +/_ mice but not WT control mice.
  • mice show significantly changed relative power in delta, alpha, and gamma frequencies compared to CDKL5+/- PBS treated mice.
  • FIG. 16A shows that BIP-TATk28k-hCDKL5 reduces abnormal gamma frequency power in aged female CDKL5 +/_ mice during the dark to light cycle transition (6am-7am).
  • FIG. 16B shows that BIP-TATk28k-hCDKL5 reduces abnormal gamma frequency power in aged female CDKL5 +/_ mice during the middle of the light cycle (12pm - 2pm).
  • CDKL5 +/_ female mice we aged CDKL5 +/_ female mice and controls to 6-7 months of age when they are known to exhibit epileptic spasms.
  • CDKL5 +/_ female mice and controls were subject to intracerebroventricular injection with PBS or AAVhu68-CBH-BIP-TATk28-hCDKL5co2 vector at a dose of 1 x 10 11 (lei 1) GC/mouse.
  • an intracranial electroencephalogram (EEG) implant was cemented onto the head, and at one month after injection the electrical activity of the left and right cortices was recorded over a 48-hour period. EEG activity was analyzed between 12 pm and 2pm in the middle of the light cycle where mice are less active and sleep frequently, and between 6am and 7am.
  • 16B shows that summed delta (0-4 Hz) power is significantly decreased in PBS treated CDKL5 +/_ mice compared with PBS treated wild type controls (12pm - 2pm). The fraction of summed gamma power in CDKL5 +/_ mice was significantly increased relative to wild type PBS controls. BIP-TATk28-hCDKL5 vector treatment significantly decreased pro-seizure gamma power.
  • FIG. 16C shows that BIP-TATk28k-hCDKL5 reduces abnormalities in gamma frequency power in CDKL5 +/_ mice at the dark to light transition (6am-7am window).
  • FIG. 16C shows representative 1-hour long electrocardiogram traces from PBS and BIP-TATk28- hCDKL5 vector treated control and CDKL5 +/_ mice.
  • PBS treated CDKL5 +/_ mice show substantial increase (hotter colors) in gamma (25 -50 Hz) frequency power compared with PBS treated controls.
  • BIP-TATk28-hCDKL5 vector reduced gamma frequency power in CDKL5 +/_ , but not WT control mice.
  • ICV treatment in aged (>6 months) seizure-prone CDKL5 +/_ mice shows a positive effect on abnormal EEG activity during the transition from active to inactive/sleep (6-7 am) and other times of day analyzed.
  • Expression of BIP-TATk28-hCDKL5 is strongest in the hippocampus with partial expression in the cortex, striatum, and thalamus close to the injection site.
  • BIP-TATk28-CDKL5 mitigates electrical abnormalities in the brains of aged CDKL5 +/_ mice.
  • aged heterozygous CDKL5 females showed e in delta and gamma frequency band power.
  • BIP-TATk28-hCDKL5 ameliorated established EEG abnormalities in aged and seizure prone CDKL5 +/_ female mice.
  • BIP- TATk28-hCDKL5 improved the power of a subset of delta frequencies, and also decreased seizure-associated increase in gamma power.
  • BIP-TATk28-hCDKL5 improved the power of a subset of delta and theta frequencies, and also decreased pro-seizure increase in gamma power. It is important to note that gamma power increased over time with age in CDKL5 +/_ acting as a potential biomarker of disease severity and progression. Furthermore, we observed that overexpression of BIP-TATk28-hCDKL5 isoform 1 on wild type background did not significantly impact summed frequency band power, and did not significantly impact behavior (open field (OF), nesting behavior (NB), home caged (HC)) either.
  • OF open field
  • NB nesting behavior
  • HC home caged
  • RNA protein codetection kit from Advanced Cell Diagnostics according to the manufacturer’s instructions.
  • S957D sheep anti-CDKL5 antibody
  • PPU MRC Protein Pohsoporylation and Ubiquitylation Unit
  • RNAscope probe to BIP- TATk28-hCDKL5 (Advanced Cell Diagnostics). Dual labeled sections were scanned and the numbers of cells in each section positive for Cdkl5 mRNA and/or protein were quantitated using Visiopharm® software.
  • mice were singly housed in a new cage with bedding and the cage then placed in the open field system within an array of infrared cross beams. Mice were allowed to freely roam their cage for 30 minutes during which the number of horizontal beam breaks (a measure of locomotor activity) or total ambulatory distance, was automatically recorded then analyzed.
  • To test nest building ability singly housed mice were transferred a new cage with a pre-weighed square nestlet. After 24 h, mice were returned to their home cage and the quality of the nest was scored on a scale of 1 to 5 according to Deacon R.M. Digging and marble burying in mice: simple methods for in vivo identification of biological impacts.
  • Electroencephalogram (EEG) recordings of brain electrical activity were performed using a 3 channel-video tethered electroencephalography system (Pinnacle Technology Inc). Trace analyses were performed using PinnacleTM software and Labchart (AD Instruments).
  • An intracranial EEG implant was cemented onto the head of untreated 3-4 month old or 6-7 month-old control and Cdkl5 +/ " mice treated with PBS or AAVhu68-CBH-BIP-TATk28- hCDKL5.
  • the implant was cemented onto the heads of treated mice one month after injection.
  • the implant containing two recording electrodes and the electrical activity of the left and right cortices was recorded over a 48-hour period.
  • EEG activity was analyzed during different time periods during the dark and light cycle including: the dark to light transition where mice transition from active to less active and sleep states (6am-7am), the middle of the light cycle (12 pm and 2pm), the beginning of the dark cycle (6pm -7 pm) and the middle of the dark cycle (12 am to 2 am) where mice are more active and sleep less.
  • Example 4 Evaluation of the pharmacology and toxicity of an AAVhu68.CBH.BIP- TATk28-CDKL5-1.SV40 vector delivered into the cisterna magna of rhesus macaques
  • the readouts of the study included: BW, Clinpathology with coag., NAb/Bab, peripheral blood mononuclear cells (PBMCs), cerebrospinal fluid (CSF) & serum biomarkers (DRG), Neuro-exam, NCV; histopathology: DRG toxicity, H&E, CDKL5 ISH, CDKL5 IHC; biochemical: western blot; bio-distribution; neurofilament light chain (Nfl).
  • Sample collected for analysis included baseline, day 0 (DO), D7, D15, D28, D35/36. Furthermore, necropsies were completed on D35/36.
  • subject 191410 showed mild lymphocytic pleocytosis of D15 (animal stable).
  • D22 showed normal CSF values, which indicated a resolved mild pleocytosis from D14, and showed signs of doing well cage-side. No D22 clinical chemical analysis was performed due to low serum volume.
  • CSF sample indicated signs of mild lymphocytic pleocytosis (similar to D15), with normal cbc/chem analysis.
  • D36 CSF sample indicated to be normal, subdural hematoma was visible at the spinal cord and dorsal surface of cerebellum.
  • subject 171164 showed mild lymphocytic pleocytosis on D7 on CSF readout, but observed to be doing well on cage-side observation, and appeared to be clinically normal. No notable observations were observed at D15. On D22, moderate mononuclear/lymphocytic pleocytosis was indicated in CSF results. On D28, moderate lymphocytic pleocytosis was indicated on CSF analysis (216 WBC/pL, 98% lymphocytes, approximately doubled since D22), while CBC/chem was normal with clinically normal observation. On D36, reddening along margins of lungs was observed, with a mild lymphocytic pleocytosis, values improved from D28 (216 to 37 WBC).
  • subject HS 1602026 showed mild neutrophilic leukocytosis (14. Ik WBC, 10k neutrophils) on bloodwork D7. Similar pattern was observed at baseline. There were no signs of systemic infection, with cage-side observation and CSF were observed to be normal as well. On DI 5, mild lymphocytic pleocytosis was indicated in collected sample (no blood work abnormalities, animal stable). On D22, mild mononuclear pleocytosis was indicated on CSF, slightly higher than D15 results (4 WBC on 5/25 to 20 WBC on 6/1).
  • FIG. 20 shows biodistribution results for peripheral tissue, brain, dorsal root ganglion (DRG), spinal cord (SC), plotted as GC/diploid cell. The values are summarized in tables 1A, IB, and 1C below.
  • FIG. 21 shows RNA biodistribution for select tissues of peripheral organs, brain, DRG and SC. The values are summarized in tables 2A, 2B, and 2C below.
  • hCDKL5 pool A-l includes the Bip signal, TATk28 signal and peptide linker, while pool A, B, C, and D are for the remainder of the hCDKL5 sequence and excludes the BIP signal, TATK28 signal, and peptide linker.
  • hCDKL5 pool A-l comprised peptides 1-11 to include BiP signal, Tatk28 signal and peptide linker.
  • hCDKL5 pool A comprised peptides 12-50 to exclude BiP signal, Tatk28 signal and peptide linker.
  • FIG. 22 shows summarized results of the ELISPOT analysis (Tables 3A and 3B) for AAVhu68, plotted as SFU per 10 6 (million) cells.
  • FIG. 23 shows summarized results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5, plotted as SFU per 10 6 (million) cells. These results indicate some liver transduction with AAVhu68 and hCDKL5 expression following ICM administration.
  • FIG. 24A shows results of the ELI SPOT analysis (Table 3 A) for AAVhu68 pool A, plotted as SFU per 10 6 (million) cells.
  • FIG. 24B shows results of the ELISPOT analysis (Table 3A) for AAVhu68 pool B, plotted as SFU per 10 6 (million) cells.
  • FIG. 24C shows results of the ELISPOT analysis (Table 3A) for AAVhu68 pool C, plotted as SFU per 10 6 (million) cells.
  • FIG. 24D shows results of the ELISPOT analysis (Table 3 A) for AAVhu68 baseline PBMC (for subjects 171164, 191410, HS1602026 as analyzed with DMSO, Pool A, Pool B, and Pool C), plotted as SFU per 10 6 (million) cells.
  • FIG. 24E shows results of the ELISPOT analysis (Table 3A) for AAVhu68 D35/36 PBMC (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A, Pool B, and Pool C), plotted as SFU per 10 6 (million) cells.
  • FIG. 24E shows results of the ELISPOT analysis (Table 3A) for AAVhu68 D35/36 PBMC (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A, Pool B,
  • FIG. 24F shows results of the ELISPOT analysis (Table 3A) for AAVhu68 spleen sample (for subjects 171164, 191410, HS1602026 as analyzed with DMSO, Pool A, Pool B, and Pool C), plotted as SFU per 10 6 (million) cells.
  • FIG. 24G shows results of the ELISPOT analysis (Table 3A) for AAVhu68 liver sample (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A, Pool B, and Pool C), plotted as SFU per 10 6 (million) cells.
  • FIG. 3A shows results of the ELISPOT analysis for AAVhu68 spleen sample (for subjects 171164, 191410, HS1602026 as analyzed with DMSO, Pool A, Pool B, and Pool C), plotted as SFU per 10 6 (million) cells.
  • 24H shows results of the ELISPOT analysis (Table 3 A) for AAVhu68 lymph node sample (for subjects 171164, 191410, HS1602026 as analyzed with DMSO, Pool A, Pool B, and Pool C), plotted as SFU per 10 6 (million) cells.
  • FIG. 25 A shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool A-l, plotted as SFU per 10 6 (million) cells.
  • FIG. 25B shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool A, plotted as SFU per 10 6 (million) cells.
  • FIG. 25C shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool B, plotted as SFU per 10 6 (million) cells.
  • FIG. 25 A shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool A-l, plotted as SFU per 10 6 (million) cells.
  • FIG. 25B shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool A-l, plotted as SFU per 10 6 (million) cells.
  • 25D shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool C, plotted as SFU per 10 6 (million) cells.
  • FIG. 25E shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 PBMC pool D, plotted as SFU per 10 6 (million) cells.
  • FIG. 25F shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 baseline PBMC sample (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool B, Pool C, Pool D peptides), plotted as SFU per 10 6 (million) cells.
  • 25 G shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 D35/D36 PBMC sample (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool B, Pool C, Pool D peptides), plotted as SFU per 10 6 (million) cells.
  • FIG. 25H shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 spleen sample (for subjects 171164, 191410, HS1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool B, Pool C, Pool D peptides), plotted as SFU per 10 6 (million) cells.
  • FIG. 3A and 3B shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 spleen sample (for subjects 171164, 191410, HS1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool
  • FIG. 251 shows results of the ELISPOT analysis (Tables 3A and 3B) for hCDKL5 liver sample (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool B, Pool C, Pool D peptides), plotted as SFU per 10 6 (million) cells.
  • FIG. 25J shows results of the ELISPOT analysis (Tables 3 A and 3B) for hCDKL5 lymph node sample (for subjects 171164, 191410, HS 1602026 as analyzed with DMSO, Pool A-l, Pool A, Pool B, Pool C, Pool D peptides), plotted as SFU per 10 6 (million) cells. Overall, these results indicate a transgene specific T cell responses seen in all animals. And which are only against peptide pool A- 1 that contain the TAT tag.
  • NMV results peripheral nerve function
  • FIG. 26A shows measured velocity in left medial nerve.
  • FIG. 26B shows measured velocity in right medial nerve.
  • FIG. 26C shows NP amplitude in left medial nerve.
  • FIG. 26D shows NP amplitude in right medial nerve.
  • FIG. 26E shows PP amplitude in left medial nerve.
  • FIG. 26F shows PP amplitude in right medial nerve.
  • FIG. 26G shows measured velocity in left sural nerve.
  • FIG. 26H shows measured velocity in right sural nerve.
  • FIG. 261 shows NP amplitude in left sural nerve.
  • FIG. 26J shows NP amplitude in right sural nerve.
  • FIG. 26K shows PP amplitude in left sural nerve.
  • FIG. 26L shows PP amplitude in right sural nerve.
  • Velocity is an indication of how myelinated the neurons are, i.e., if slowed velocity then demyelinating lesion.
  • Amplitude is an indication of how strong the signal from the nerve is, i.e., if lower amplitude then less neurons contributing to the signal propagation of the nerve, indication of axonopathy.
  • Tables 4A and 4B shows a summary of quantification of expression of hCDKL5 as analyzed by IHC microscopy of spinal cord tissue samples (SC-C: cervical; SC- T: thoracic; SC-L: lumbar).
  • Tables 5 A and 5B shows a summary of quantification of expression of hCDKL5 as analyzed by ISH microscopy of DRG tissue samples (DRG-C: cervical; DRG-T : thoracic;
  • DRG-L lumbar
  • DRG-S sacral

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Abstract

La présente invention concerne un virus adéno-associé recombiné (rAAV) possédant une capside AAV et un génome de vecteur comprenant une cassette d'expression de CDKL5 humain (hCDKLK5). La présente invention concerne également un système de production servant à produire le rAAV, une composition pharmaceutique comprenant le rAAV, et un procédé pour traiter un sujet souffrant de CDD, ou atténuer les symptômes d'une CDD, ou retarder la progression d'une CDD par l'administration d'une quantité efficace de rAAV à un sujet en ayant besoin.
PCT/US2022/079025 2021-11-01 2022-11-01 Compositions utiles pour traiter les troubles dus à la déficience en cdkl5 (cdd) WO2023077143A1 (fr)

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Citations (2)

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US20190241633A1 (en) * 2016-05-04 2019-08-08 Curevac Ag Rna encoding a therapeutic protein
WO2021087282A1 (fr) * 2019-10-30 2021-05-06 Amicus Therapeutics, Inc. Protéines cdkl5 recombinées, thérapie génique et procédés de production

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190241633A1 (en) * 2016-05-04 2019-08-08 Curevac Ag Rna encoding a therapeutic protein
WO2021087282A1 (fr) * 2019-10-30 2021-05-06 Amicus Therapeutics, Inc. Protéines cdkl5 recombinées, thérapie génique et procédés de production

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