Adeno-associated virus vector for gene transfer to nervous system cells
The present invention provides a means for transferring a therapeutic gene of interest into a nervous system cell by a highly-efficient and simpler means. More specifically, the present invention provides a recombinant vector that uses an adeno-associated virus (AAV), a method for manufacturing the recombinant vector, and a method for using the recombinant vector. More specifically, recombinant adeno-associated virus virions, which are capable of passing through the brain-brain barrier, for transferring a therapeutic genes of interest into a nervous system cell in a highly-efficient manner, a drug composition containing the recombinant adeno-associated virus virions, a method for manufacturing the recombinant adeno-associated virus virions, and a kit or the like are provided.
1. A method of delivering a therapeutic gene of interest to a nerve cell in a brain of a living subject, comprising:
providing a recombinant adeno-associated virus (rAAV) vector, and
peripherally administering said rAAV vector to a subject;
wherein said rAAV vector is configured to pass through an adult, fetus or neonate blood-brain barrier to deliver a therapeutic gene of interest to a nerve cell in a brain of a living subject, and comprises:
(a) a capsomere which comprises a protein comprising the amino acid sequence of SEQ ID NO: 8 or 12, wherein the protein is capable of forming a virus virion, and
(b) a polynucleotide packaged in said capsomere, the polynucleotide comprising a nervous system cell-specific promoter sequence and a nucleotide sequence as a gene operably linked to the promoter sequence, wherein the promoter sequence is selected from the group consisting of (i) a synapsin I promoter sequence, (ii) a myelin basic protein promoter sequence, and (iii) a L7 promoter sequence that is a cerebellar Purkinje cell specific promoter sequence;
said recombinant adeno-associated virus vector is not self-complementary (sc); and
said peripherally administering is not to blood vessels in the head of the subject.
2. The method according to claim 1 , wherein the 5′ end of the polynucleotide comprises a 5′ end-inverted terminal repeat (ITR) sequence derived from adeno-associated virus serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4) or serotype 9 (AAV9), and the 3′ end of the polynucleotide comprises a 3′ end-ITR sequence derived from AAV1, AAV2, AAV3, AAV4, or AAV9.
3. The method according to claim 1 , wherein the 5′ and 3′ ends of the polynucleotide comprise the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively.
4. The method according to claim 1 , wherein the polynucleotide has a full length of 2 to 6 kb and is a single stranded DNA which is a sense strand or an antisense strand.
5. The method according to claim 4 , wherein the promoter sequence comprises the polynucleotide of SEQ ID NO: 23 or SEQ ID NO: 24.
6. The method according to claim 4 , wherein the nucleotide sequence operably linked to the promoter sequence encodes a protein selected from the group consisting of an antibody, a nerve growth factor (NGF), a growth factor, an acidic fibroblast growth factor (aFGF), a basic fibroblast growth factor (bFGF), a glial cell line-derived neurotrophic factor (GDNF), an aromatic amino acid decarboxylase, and an amyloid 13 degrading protease that is Neprilysin.
7. The method according to claim 4 , wherein the nucleotide sequence operably linked to the promoter sequence expresses dsRNA, siRNA, shRNA or miRNA to downregulate expression of an aromatic amino acid decarboxylase or α-synuclein.
8. The method according to claim 6 , wherein the antibody is an antibody that binds to aggregated amyloid 13 protein.
9. The method according to claim 1 , wherein the living subject is human.
10. The method according to claim 1 , wherein the capsomere which comprises the protein comprises the amino acid sequence of SEQ ID NO: 8.
11. The method according to claim 1 , wherein the capsomere which comprises the protein comprises the amino acid sequence of SEQ ID NO: 12.
12. The method according to claim 1 , wherein the promoter sequence is the synapsin I promoter sequence.
13. The method according to claim 1 , wherein the promoter sequence is the myelin basic protein promoter sequence.
14. The method according to claim 1 , wherein the peripherally administering is performed by intraarterial administration, intrapericardial administration, intramuscular administration, or umbilical intravascular administration.
15. A method of treating a subject with a neurological disorder, an inborn error of metabolism in a neuron, or a demyelinating disorder in a neuron, comprising delivering a therapeutic gene of interest by the method of claim 1 .
16. The method according to claim 15 , wherein the neurological disorder, the inborn error of metabolism, or the demyelinating disorder is selected from the group consisting of: Parkinson's disease, Alzheimer's disease, triplet repeat disease, prion disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, channel disease, epilepsy; Wilson's disease, peroxisome disease; and multiple sclerosis.