JPWO2020097417A5

JPWO2020097417A5 -

Info

Publication number: JPWO2020097417A5
Application number: JP2021524359A
Authority: JP
Publication date: 2022-11-16

Claims

A non-viral capsid-free DNA vector (ceDNA vector) with covalently closed ends, said ceDNA vector being operably positioned between two flanking symmetrical inverted terminal repeats ( symmetric ITRs) a non-viral capsid-free DNA vector with covalently closed ends ( ceDNA vector ).

2. The ceDNA vector of claim 1, wherein said symmetric ITR is synthetic.

4. The ceDNA vector of any one of the preceding claims, wherein said ITRs are selected from the group consisting of said ITRs listed in Table 4.

each of said symmetric ITRs is deleted, inserted and/or substituted in at least one of said ITR regions selected from A, A', B, B', C, C', D and D' 11. The ceDNA vector of any one of the preceding claims, which is modified by

4. Said deletion, insertion and/or substitution results in deletion of all or part of the stem-loop structure formed by said A, A', B, B'C, or C' regions. The ceDNA vector described in .

each of said symmetric ITRs is modified by deletion, insertion and/or substitution resulting in the deletion of all or part of the stem-loop structure formed by said B and B' regions ;
each of said symmetric ITRs is modified by deletion, insertion and/or substitution resulting in the deletion of all or part of the stem-loop structure formed by said C and C'regions;
each of the symmetric ITRs has all or deletion of a portion of the stem-loop structure formed by the B and B' regions and/or a portion of the stem-loop structure formed by the C and C'regions; modified by resulting deletions, insertions, and/or substitutions; or
each of said symmetric ITRs is modified by deletion of all of the stem-loop structure formed by said B and B' regions or all of the stem-loop structure formed by said C and C'regions;
The ceDNA vector according to claim 4 or 5.

A region in which each of the symmetric ITRs includes a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions in the wild-type ITR. contains a single stem-loop structure ;
A region in which each of the symmetric ITRs includes a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions in the wild-type ITR. contains a single stem and two loops; or
A region in which each of the symmetric ITRs includes a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions in the wild-type ITR. contains a single stem and a single loop ,
A ceDNA vector according to any one of the preceding claims .

The ceDNA vector according to any one of the preceding claims, comprising an aptamer.

11. The ceDNA vector of any one of the preceding claims , comprising at least one regulatory switch.

A non-viral capsid-free DNA vector (ceDNA vector) with covalently closed ends, said ceDNA vector being operable between two flanking wild-type inverted terminal repeats ( WT-ITR) A non-viral capsid-free DNA vector (ceDNA vector) with covalently closed ends comprising at least one arranged heterologous nucleic acid sequence, said ceDNA vector comprising at least one regulatory switch.

11. The ceDNA vector of claim 10 , wherein said WT-ITR is a symmetric WT-ITR or a substantially symmetric WT-ITR.

11. The ceDNA vector of any one of the preceding claims, wherein said symmetric ITR is derived from a virus selected from the group consisting of parvovirus, dependovirus and adeno-associated virus (AAV).

13. The ceDNA of claim 12 , wherein said symmetric ITR is derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. vector.

11. The ceDNA vector of any one of the preceding claims, wherein the vector is within a lipid nanoparticle (LNP) .

wherein said ceDNA vector is (a) subjected to a population of insect cells carrying a ceDNA expression construct in the presence of at least one Rep protein under conditions effective to induce production of said ceDNA vector in said insect cells; and (b) isolating the ceDNA vector from the insect cells. , a ceDNA vector according to any one of the preceding claims .

16. The ceDNA vector of claim 15, wherein said insect cell expresses at least one Rep protein.

A ceDNA expression construct encoding a ceDNA vector according to any one of the preceding claims .

A host cell comprising the ceDNA expression construct of claim 17 .

19. The host cell of claim 18 , which expresses at least one Rep protein.

20. The host cell of claim 19 , wherein said at least one Rep protein is derived from a virus selected from the group consisting of parvovirus, dependovirus and adeno-associated virus (AAV).

21. The method of claim 20 , wherein said at least one Rep protein is from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. host cells.

A host cell according to any one of claims 18-21 , which is an insect cell.

23. The host cell of claim 22 , wherein said insect cell is an Sf9 cell.

24. A method of producing a ceDNA vector, comprising: (a) incubating the host cell of any one of claims 18-23 under conditions effective and for a time sufficient to induce production of said ceDNA vector. and (b) isolating said ceDNA from said host cell.

17. The ceDNA vector of any one of claims 1 , 2, 4-5, 7 and 9-16 for use in a method for treating, preventing, ameliorating, monitoring or diagnosing a disease or disorder in a subject A pharmaceutical composition comprising

26. The pharmaceutical composition of claim 25 , wherein said at least one heterologous nucleic acid sequence, when transcribed or translated, modifies the abnormal amount , function or activity of an endogenous protein in said subject.

said at least one heterologous nucleic acid sequence encodes or comprises a nucleotide molecule selected from the group consisting of RNAi, siRNA, miRNA, lncRNA, aptamers and antisense oligonucleotides or polynucleotides ; or
said at least one heterologous nucleic acid sequence encodes a protein; or
said at least one heterologous nucleic acid sequence encodes an agonist or antagonist of an endogenous protein or pathway associated with said disease or disorder; or
said at least one heterologous nucleic acid sequence encodes an antibody ;
26. A pharmaceutical composition according to claim 25 .

said disease or disorder is
a metabolic disease or disorder selected from the group consisting of diabetes, lysosomal storage disorders, mucopolysaccharide disorders, urea cycle diseases or disorders, and glycogen storage diseases or disorders; wherein said lysosomal storage disorder is Gaucher disease, Pompe disease, is selected from the group consisting of metachromatic leukodystrophy (MLD), phenylketonuria (PKU) and Fabry disease; said urea cycle disease or disorder is ornithine transcarbamylase (OTC) deficiency; said mucopolysaccharidosis is a metabolic disease or disorder selected from the group consisting of Sly's Syndrome, Hurler's Syndrome, Scheie's Syndrome, Hurler-Scheie's Syndrome, Hunter's Syndrome, Sanfilippo's Syndrome, Morquio's Syndrome and Marlotauramie Syndrome;
A CNS disease or disorder , Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy , Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma due to spinal cord or head injury, Tay-Sachs disease, Leschenyan's disease, epilepsy, cerebral infarction, psychiatric disorders, schizophrenia, drug dependence neurosis, psychosis, dementia, paranoia, attention deficit disorder, sleep disorders, pain disorders, eating or weight disorders, and cancers and tumors of the CNS;
an ocular disease or disorder selected from the group consisting of an ophthalmic disorder involving the retina, the posterior thalamic pathway and/or the optic nerve ; retinopathy, macular degeneration including age-related macular degeneration, geographic atrophy and vascular or "exudative" macular degeneration, glaucoma, uveitis, retinitis pigmentosa, Stargardt's disease, Leber congenital amaurosis (LCA), Usher's syndrome, Pseudoxanthoma elasticum (PXE), X-linked retinitis pigmentosa (XLRP), X-linked retinoschisis (XLRS), total choroidal atrophy, Leber's hereditary optic atrophy (LHON), color blindness, cone-rod an ocular disease or disorder selected from the group consisting of degeneration, Fuchs corneal endothelial degeneration, diabetic macular edema, and ocular cancers and tumors;
a blood disease or disorder selected from the group consisting of hemophilia A, hemophilia B, thalassemia, anemia and blood cancer ;
a liver disease or disorder selected from the group consisting of progressive familial intrahepatic cholestasis (PFIC) and liver cancer, and a tumor;
cystic fibrosis; and
28. Any one of claims 25-27 selected from the group consisting of immune diseases or disorders, infectious diseases, muscle diseases or disorders, cancer, and diseases or disorders based on abnormal levels and/or functions of gene products. Pharmaceutical composition as described.

The pharmaceutical composition according to any one of claims 25-28, wherein said ceDNA vector further comprises an aptamer.

A pharmaceutical composition comprising the ceDNA vector of any one of claims 1 , 2, 4-5, 7 and 9-16 for use in a method for delivering a therapeutic protein to a subject , , a pharmaceutical composition , wherein said at least one heterologous nucleic acid sequence encodes a therapeutic protein.

A kit comprising the ceDNA vector of any of claims 1 , 2, 4-5, 7 and 9-16 and a nanocarrier packaged in a container with instructions for use .