WO2023018674A1 - Détermination de la puissance de transduction des gènes dans les cellules de type neuronal - Google Patents

Détermination de la puissance de transduction des gènes dans les cellules de type neuronal Download PDF

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WO2023018674A1
WO2023018674A1 PCT/US2022/039763 US2022039763W WO2023018674A1 WO 2023018674 A1 WO2023018674 A1 WO 2023018674A1 US 2022039763 W US2022039763 W US 2022039763W WO 2023018674 A1 WO2023018674 A1 WO 2023018674A1
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protein
cells
disease
ceroid
disease type
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PCT/US2022/039763
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Jacob CAIN
Sean Sullivan
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Amicus Therapeutics, Inc.
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Publication of WO2023018674A1 publication Critical patent/WO2023018674A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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

  • the present invention generally relates to assays for assessing adeno-associated virus (AAV) vectors.
  • AAV adeno-associated virus
  • Genetic disorders arise via heritable or de novo mutations occurring in gene coding regions of the genome.
  • such genetic disorders are treated by administration of a protein encoded by the gene mutated in the individual having the genetic disorder.
  • Such treatment has challenges however, as administration of the protein does not always result in the protein reaching the organs, cells, or organelle where it is needed.
  • this treatment also often requires frequent infusions, which are not needed with gene therapy, where a single treatment can offer lasting relief. Therefore, gene therapy has the potential to offer improved results over currently available treatments for genetic disorders.
  • Various aspects of the present invention relate to a method for determining the transduction efficiency of an AAV vector comprising a transgene.
  • the method comprises culturing neuron-like cells in a culture media, transducing the neuron-like cells with the AAV vector comprising the transgene, and measuring the proportion of the transduced cells expressing protein encoded by the transgene.
  • the neuron-like cells comprise one or more of primary mouse neurons, human fibroblasts, HEK-293, Lec2, HT-22, C8-Dla, Neuro2A, SH- SY5Y, neural progenitor cells, or a variant thereof.
  • the neural progenitor cells are grown as adherent neural stem cells or neurosphere cells.
  • the neuron-like cells comprise one or more of HT-22, C8-Dla, Neuro2A, neurosphere cells, or a variant thereof.
  • the neuron-like cells are derived from mouse. In one of more embodiments, the neuron-like cells are derived from mouse C57BL/6 cells, or a variant thereof.
  • the culture media comprises neural stem cell media.
  • the transgene comprises a reporter gene such as GFP.
  • the transgene comprises a sequence encoding a therapeutic protein.
  • the therapeutic protein is capable of replacing a defective or deficient protein associated with a genetic disorder in a subject having the genetic disorder.
  • the genetic disorder is a lysosomal storage disorder.
  • the genetic disorder is selected from the group consisting of aspartylglucosaminuria, Batten disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteau-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type Cl, Niemann-Pick disease type C2, Schindler disease type I, Schindler disease type
  • the therapeutic protein comprises an enzyme.
  • the enzyme is selected from the group consisting of alphagalactosidase, P-galactosidase, P-hexosaminidase, galactosylceramidase, arylsulfatase, P- glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme, iduronidase, acetyl-CoA:alpha- glucosaminide N-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparan N- sulfatase, N-acetyl-a-D-glucosaminidase, iduronate-2-sulfatas
  • the therapeutic protein is a Batten-related protein.
  • the Batten-related protein is a ceroid-lipofuscinosis neuronal protein.
  • the ceroid-lipofuscinosis neuronal protein is ceroid- lipofuscinosis neuronal protein 1, ceroid- lipofuscinosis neuronal protein 2, ceroidlipofuscinosis neuronal protein 3, ceroid- lipofuscinosis neuronal protein 4, ceroidlipofuscinosis neuronal protein 5, ceroid- lipofuscinosis neuronal protein 6, ceroidlipofuscinosis neuronal protein 7, ceroid- lipofuscinosis neuronal protein 8, ceroid- lipofuscinosis neuronal protein 9, ceroid-lipofuscinosis neuronal protein 10, ceroidlipofuscinosis neuronal protein 11, ceroid- lipofuscinosis neuronal protein 12, ceroidlipofuscinosis neuronal protein 13, or ceroid
  • the transgene further comprises a translation initiation sequence. In one or more embodiments, the trans gene further comprises a linker sequence encoding a linker peptide between a nucleic acid sequence encoding a fusion protein and the therapeutic protein.
  • the AAV vector is a self-complementary AAV (scAAV). In one or more embodiments, the AAV vector is a single-stranded AAV (ssAAV). In one or more embodiments, the AAV vector is a recombinant AAV (rAAV). In one or more embodiments, the AAV vector comprises AAV9, AAVHu68, AAV2, AAV8, AAV5, or a variant thereof. In one or more embodiments, the AAV vector further comprises inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • the neuron-like cells prior to transduction, are treated with neuraminidase type III.
  • the proportion of transduced cells expressing transgene protein is determined by using immunocytochemistry, mRNA, or protein analysis.
  • the immunocytochemistry comprises flow cytometry.
  • the method further comprises quantifying the expressed transgene protein in the transduced cells.
  • the expressed transgene protein is quantified by immunocytochemistry and/or protein analysis.
  • Figure 1 shows the relative transduction efficiency for an scAAV9-CLN6 vector in neurosphere, C8Dla, Neuro2A and HT-22 cells.
  • the cells were transduced at 3xl0 4 MOI.
  • immunocytochemistry was performed on the cells, where the cells expressing transgene protein were labelled with a primary antibody, Rb anti-CLN6, and subsequently labelled with a secondary antibody, Donkey anti-Rabbit IgG(H+L)-Alexa Fluor Plus 647.
  • the potency of scAAV9-CLN6 vector for transduced cells expressing transgene protein was determined by flow cytometry.
  • Various embodiments of the present invention relate to a potency assay for determining infectivity for AAV vectors in neuron-like cells.
  • the method is used to determine transduction efficiency and/or quantify the amount of transgene expression.
  • neuron-like cells refers to any type of cells that can be used to mimic the functioning of neurons or understand the functioning of neurons.
  • Non-limiting, exemplary neuron-like cells include primary mouse neurons, human fibroblasts, HEK-293, Lec2, HT-22, C8-Dla, Neuro2A, SH-SY5Y, neural progenitor cells, or a variant thereof.
  • neuralsphere cells refers to neural stem cells, neural stem cell cluster, or their progeny.
  • therapeutic protein refers to a protein that is capable of replacing a defective or deficient protein associated with a genetic disorder in a subject having a genetic disorder.
  • Non-limiting, exemplary genetic disorders include aspartylglucosaminuria, Batten disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteau-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann- Pick disease type B, Niemann-Pick disease type Cl, Niemann- Pick disease type C2, Schindler disease type I, Schindler disease type II, adenosine deaminase severe combined immunodeficiency, chronic granulomatous disease, CDKL
  • the therapeutic protein could also be an enzyme.
  • Non-limiting, exemplary enzymes include alpha-galactosidase, P-galactosidase, P-hexosaminidase, galactosylceramidase, arylsulfatase, P-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme, iduronidase, acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycan alpha- L-iduronohydrolase, heparan N-sulfatase, N-acetyl-a-D-glucosaminidase, iduronate-2- sulfatase, galactosamine- 6- sulfate
  • ceroid lipofuscinosis neuronal proteins refers to proteins implicated in Batten disease such as ceroid lipofuscinosis neuronal proteins.
  • Non-limiting, exemplary ceroid lipofuscinosis neuronal proteins includes ceroid-lipofuscinosis neuronal protein 1, ceroid- lipofuscinosis neuronal protein 2, ceroid- lipofuscinosis neuronal protein 3, ceroidlipofuscinosis neuronal protein 4, ceroid- lipofuscinosis neuronal protein 5, ceroidlipofuscinosis neuronal protein 6, ceroid-lipofuscinosis neuronal protein 7, ceroidlipofuscinosis neuronal protein 8, ceroid-lipofuscinosis neuronal protein 9, ceroidlipofuscinosis neuronal protein 10, ceroid- lipofuscinosis neuronal protein 11, ceroidlipofuscinosis neuronal protein 12, ceroid-lipofuscinosis neuronal protein 13, and ceroidlipofuscinosis neuronal protein 14.
  • the potency of the AAV vector comprising the transgene is determined by culturing neuron- like cells, treating the neuron- like cells with neuraminidase type III, transducing the neuraminidase treated cells by incubating with the AAV vector comprising the transgene, and determining proportion of transduced cells expressing protein encoded by the transgene.
  • the neuron-like cells can be prepared for the assay using standard culture techniques, e.g., culturing, trypsinizing, quenching of trypsinization, and determining cell culture density.
  • the cells are cultured in cell culture media at 37°C and 5% CO2.
  • the cells are then treated with trypsin until the cells detach. Typically, trypan blue is used to stain dead cells.
  • a hemocytometer may be used to calculate the density of viable cells.
  • AAV Adeno-associated virus
  • the AAV vector is an rAAV.
  • AAV vectors typically include one or more inverted terminal repeat (ITR) sequences, a replication (Rep) gene sequence, and a capsid (Cap) gene sequence.
  • ITR inverted terminal repeat
  • Rep and Cap sequences may be included in the same plasmid (in cis)' , or may be provided in separate plasmids (in trans).
  • the capsid may be derived from the same serotype as the ITR sequences, or the AAV vector can be a hybrid vector utilizing ITR sequences and capsids derived from different AAV serotypes.
  • AAV of any serotype is contemplated to be used.
  • the serotype of the viral vector used in certain embodiments is selected from the group consisting of AAV9, AAVHu68, AAV2, AAV8, AAV5, and a variant thereof.
  • the transgene comprises a reporter gene, e.g., green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • the transgene comprises a sequence encoding a therapeutic protein.
  • the transgene comprises a sequence encoding a therapeutic protein, wherein the therapeutic protein is capable of replacing a defective or deficient protein associated with a genetic disorder in a subject having the genetic disorder, e.g., aspartylglucosaminuria, Batten disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay Sachs disease, Sandhoff disease, metachomatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteau-Lamy disease, Sly disease, Niemann- Pick disease type A,
  • the genetic disorder e.g., as
  • the transgene comprises a sequence encoding a therapeutic protein, wherein the therapeutic protein is an enzyme.
  • exemplary enzymes include, but are not limited to, alpha-galactosidase, P-galactosidase, P-hexosaminidase, galactosylceramidase, arylsulfatase, P-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine-generating enzyme, iduronidase, acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycan alpha- L-iduronohydrolase, heparan N-sulfatase, N-acetyl-a-D-glucosaminidase,
  • the transgene comprises a sequence encoding a therapeutic protein, wherein the therapeutic protein is a Batten-related protein, e.g., ceroid- lipofuscinosis neuronal protein 1, ceroid- lipofuscinosis neuronal protein 2, ceroidlipofuscinosis neuronal protein 3, ceroid- lipofuscinosis neuronal protein 4, ceroid lipofuscinosis neuronal protein 5, ceroid- lipofuscinosis neuronal protein 6, ceroid lipofuscinosis neuronal protein 7, ceroid- lipofuscinosis neuronal protein 8, ceroid lipofuscinosis neuronal protein 9, ceroid-lipofuscinosis neuronal protein 10, ceroid- lipofuscinosis neuronal protein 11, ceroid- lipofuscinosis neuronal protein 12, ceroid- lipofuscinosis neuronal protein 13, and ceroid-lipofuscinosis neuronal protein 14).
  • the therapeutic protein is a Batten-related protein, e.g., ceroid- lip
  • the transgene further comprises a translation initiation sequence, e.g., Kozak sequence, IRES sequence, or the like.
  • Gene transduction can be performed by known techniques.
  • gene transduction is performed by seeding, treating with neuraminidase type III, and incubation with an AAV vector comprising the transgene.
  • the cells are treated with neuraminidase type III, such as with about 50 mU/ml neuraminidase type III.
  • the cells are transduced with AAV vector comprising the transgene, such as at 1,000 to 500,000 multiplicity of infection (MOI).
  • the MOI is in the range of 2,000-10,000, 5,000-15,000, 10,000-20,000, 15,000-30,000, 20,000-50,000, 25, GOO- 75, 000, 50,000-100,000, 75,000-150,000, 100,000-200,000, 150,000-300,000, 200,000- 400,000, or 250,000-500,000.
  • Exemplary MOIs include 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000 or 500,000, or any range between these values.
  • Post infection the transduced cells are allowed to recover, such as at 37 °C and 5% CO2 for 1-10 days. In one or more embodiments, the recovery time is in the range of 1-5 days, 2-6 days, 3-7 days, 4-8 days, 5-9 days, or 6-10 days.
  • the potency can be determined by immunocytochemistry. Alternatively, the potency can be determined by mRNA or protein analysis techniques.
  • immunochemistry is used to label the transduced cells.
  • the transduced cells can be detached by trypsinization.
  • the cells aggregates can be dissociated by treating the cell suspension with cell dissociation solution.
  • the live-dead stain can be performed to measure the viability of transduced cells.
  • the cells in dissociated cell suspension can be fixed, labelled with primary antibody, and subsequently labelled with secondary antibody.
  • the secondary labelled cells are detected using flow cytometry.
  • the proportion of the transduced cells expressing the transgene is calculated.
  • the cells are analyzed to determine the quantity of expressed protein in the transduced cells. Such quantification can be determined by appropriate techniques such as immunocytochemistry or protein analysis techniques.
  • the assays described herein provide high transduction efficiency at relatively low MOIs.
  • AAVs such as AAV9 can have very low transduction efficiency without a helper AAV.
  • AAV9 can require MOIs over 100,000 in certain cells types.
  • the assays described herein allow for efficient transductions at MOIs below 100,000 such as an MOI of 30,000
  • the assay provides at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% transduction of the AAV vector in the neuron-like cells at MOIs of 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000 or 100,000.
  • Mouse cortical cells were isolated from Embryonic Day 15 mouse pups. Cells were then grown in a DMEM/F12 based media supplemented with Epidermal Growth Factor (Shenandoah 200-12), Fibroblast Growth Factor (Shenandoah 200-53), and Fetal Bovine Serum. Neurosphere cells were grown in suspension using cell culture flask not coated with a substrate.
  • Neurosphere cells which were at passage 7-15, were used for the viability count.
  • the neurosphere cells were treated with trypsin at 37°C until cells detached.
  • the trypsinization reaction was quenched by adding 37°C pre-warmed neural stem cell media. Trypan blue (Thermo Scientific 15250061) was used to stain dead cells.
  • Hemocytometer (SKC C-CHIP, disposable, Fisher Scientific 22-600-101) was used to determine the viable count for neurosphere cells.
  • the viable neurosphere cells were seeded at 350,000 viable cells/well in 24- substrate coated well plate and incubated at 37°C and 5% CO2 for about 24 hours.
  • the seeded neurosphere cells were then treated with 250 pl of neural stem cell media containing 50 mU/ml neuraminidase type III (Sigma N7885) in each well at 37°C and 5% CO2 for two hours.
  • the neuraminidase containing media was removed from each well.
  • the cells were washed with 500 pl/well of viral culture media.
  • scAAV9-CLN6 vector stock solution was prepared in virus dilution buffer such that the final MOI value would be 3xl0 4 ) (Thermo Fisher Anderson) in each well were added.
  • the cells were incubated with scAAV9-CLN6 vector at 37°C and 5% CO2. After 26 hours post infection, about 1 ml of viral recovery media was added to each well and the cells were incubated at 37°C and 5% CO2. After 74 hours post vector addition, additional 1 ml of neural stem cell media was added to each well and the cells were again incubated at 37°C and 5% CO2. After 170 hours post infection, all the media is replaced with fresh 1 ml of neural stem cell media in each well and cells were incubated at 37°C and 5% CO2. The cells are ready for downstream process after 190 hours post infection.
  • the transduced neurosphere cells above were washed with 1 ml/well of calcium/magnesium free PBS buffer 190 hours post infection. The cells were then treated with 200 pl of trypsin containing PBS buffer. Once the cells detached, the trypsinization reaction was quenched by adding 200 pl of neural stem cell media. The cell suspension was then centrifuged at 600 x g. The supernatant was discarded, and the pellet was resuspended into 100 pl of neural stem cell media. The resuspended cell suspension was then transferred into a 96- well plate.
  • the cell aggregates were dissociated by adding 100 pl of cell dissociation solution (“Accumax” from Sigma A708) to the cell suspension in each well and incubating for 30 minutes at room temperature. The cells were centrifuged at 350 x g, and the supernatant was discarded. The cells were then washed twice with 200 pl of 4°C prechilled PBS buffer, centrifuged at 350 x g, and the supernatant was discarded. Finally, the cell pellet was resuspended in residual PBS buffer and maintained at 4°C for downstream process.
  • live-dead stain (Invitrogen L34957) was mixed with the cell suspension and PBS buffer in the ration of 1:150:849 in the plate. The plate was incubated in dark at 4°C for 30 minutes. For unstained control, DMSO (Sigma D2650) was used instead of live-dead stain. The plate was then centrifuged at 350 x g. The supernatant was discarded. The stained cells were washed with 200 pl of cold wash buffer, centrifuged at 350 x g, and the supernatant was discarded. The wash step was repeated twice.
  • the cells were incubated with 150 pl/well of fixation buffer at room temperature in the container. The cells were then permeabilized with 100 pl of permeabilization buffer. The permeabilized cells were centrifuged at 1,000 x g, and the supernatant was discarded. The cells were resuspended in 200 pl of permeabilization buffer again and centrifuged at 1,000 x g. The supernatant was discarded, and the pellet was resuspended into residual permeabilization buffer. [0049] For labelling with primary antibody, the permeabilized cells were incubated with primary antibody, Rb anti-CLN6 (Amicus AB-0001, 0.99 mg/ml purified, polyclonal), containing permeabilization buffer.
  • Rb anti-CLN6 Amicus AB-0001, 0.99 mg/ml purified, polyclonal
  • the primary antibody labelled cells were centrifuged at 1,500 x g, and the supernatant was discarded. The cells were then washed with permeabilization buffer, centrifuged at 1,500 x g, and the supernatant was discarded. The wash step was repeated twice.
  • the above primary antibody labelled cells were incubated with secondary antibody (Donkey anti-Rabbit IgG(H+L), Alexa Fluor Plus 647: Invitrogen A32795) containing permeabilization buffer.
  • the secondary antibody labelled cells were centrifuged at 1,750 x g, and the supernatant was discarded.
  • the cells were then washed with permeabilization buffer, centrifuged at 1,750 x g, and the supernatant was discarded. The wash step was repeated twice.
  • the secondary antibody labelled transduced cells were resuspended into cytometry staining buffer. The samples were loaded on the flow cytometer and the readings were recorded. The transduction efficiency is calculated by using the following formula:
  • Example 1 The protocol described in Example 1 was performed in other cell types including C8Dla, Neuro2A and HT-22 cells.
  • the transduction of AAV9 at 30,000 MOI in neurospheres, C8- Dla, Neuro2A and HT-22 cells is shown in Figure 1. As can be seen, all four cells types had some transduction, with neurospheres providing the highest transduction at over 80% of the neurosphere cells being transduced.

Abstract

La présente invention concerne un procédé permettant de réaliser un dosage de puissance dans des cellules de type neuronal pour un vecteur AAV comprenant un transgène.
PCT/US2022/039763 2021-08-09 2022-08-09 Détermination de la puissance de transduction des gènes dans les cellules de type neuronal WO2023018674A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
WO2012109214A1 (fr) * 2011-02-07 2012-08-16 The University Of North Carolina At Chapel Hill Transduction ciblée de vecteurs aav
US20190203212A1 (en) * 2012-07-25 2019-07-04 Massachusetts Institute Of Technology Inducible dna binding proteins and genome perturbation tools and applications thereof
WO2019236949A1 (fr) * 2018-06-08 2019-12-12 Avexis Inc. Dosage basé sur des cellules permettant de mesurer la puissance d'un produit médicamenteux
WO2020163299A1 (fr) * 2019-02-04 2020-08-13 Research Institute At Nationwide Children's Hospital Administration de virus adéno-associé de polynucléotide cln6
WO2020210713A1 (fr) * 2019-04-10 2020-10-15 Prevail Therapeutics, Inc. Thérapies géniques pour troubles lysosomaux

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Publication number Priority date Publication date Assignee Title
WO2012109214A1 (fr) * 2011-02-07 2012-08-16 The University Of North Carolina At Chapel Hill Transduction ciblée de vecteurs aav
US20190203212A1 (en) * 2012-07-25 2019-07-04 Massachusetts Institute Of Technology Inducible dna binding proteins and genome perturbation tools and applications thereof
WO2019236949A1 (fr) * 2018-06-08 2019-12-12 Avexis Inc. Dosage basé sur des cellules permettant de mesurer la puissance d'un produit médicamenteux
WO2020163299A1 (fr) * 2019-02-04 2020-08-13 Research Institute At Nationwide Children's Hospital Administration de virus adéno-associé de polynucléotide cln6
WO2020210713A1 (fr) * 2019-04-10 2020-10-15 Prevail Therapeutics, Inc. Thérapies géniques pour troubles lysosomaux

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Title
XENIA LOJEWSKI ET AL: "Human iPSC models of neuronal ceroid lipofuscinosis capture distinct effects of TPP1 and CLN3 mutations on the endocytic pathway", HUMAN MOLECULAR GENETICS, vol. 23, no. 8, 15 April 2014 (2014-04-15), GB, pages 2005 - 2022, XP055713460, ISSN: 0964-6906, DOI: 10.1093/hmg/ddt596 *

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