Search

JP-7854715-B2 - vector

JP7854715B2JP 7854715 B2JP7854715 B2JP 7854715B2JP-7854715-B2

Inventors

  • クリストファー エドワード デニスタウン ショー
  • ヨウン ボク リー

Assignees

  • キングス・カレッジ・ロンドン

Dates

Publication Date
20260507
Application Date
20200928
Priority Date
20190927

Claims (20)

  1. (i) A human growth hormone intron 3 (hGHi3) sequence, wherein the hGHi3 sequence includes the sequence of sequence number 7; (ii) Synapsin promoter sequence; (iii) a polynucleotide coding sequence encoding progranulin (PGRN) and comprising a 5' exon splicing element (ESE); and (iv) 3' untranslated region (UTR) sequence of PGRN, An adeno-associated virus (AAV) vector containing nucleic acids.
  2. The AAV vector according to claim 1, wherein the polynucleotide coding sequence has at least 95% sequence identity with SEQ ID NO: 4.
  3. The AAV vector according to claim 1 or 2, wherein the PGRN comprises the amino acid sequence of SEQ ID NO: 16 or a variant or homolog thereof having at least 90% sequence identity with SEQ ID NO: 16 .
  4. The AAV vector according to any one of claims 1 to 3, wherein the polynucleotide coding sequence is codon-optimized.
  5. The AAV vector according to claim 4, wherein the polynucleotide coding sequence is codon-optimized for expression in humans.
  6. The AAV vector according to claim 1, wherein the ESE is part of a 5' adjacent sequence upstream of the polynucleotide coding sequence or is inserted into the 5' adjacent sequence.
  7. The AAV vector according to claim 6, wherein the ESE is a part of the 5' adjacent sequence, and the 5' adjacent sequence is derived from a wild-type polynucleotide sequence.
  8. The AAV vector according to claim 6 or 7, wherein the 5' adjacent sequence is a 5' guide sequence derived from wild-type granulin (GRN).
  9. The AAV vector according to claim 8, wherein the 5' guide sequence of the wild-type GRN contains 350 to 450 base pairs.
  10. The AAV vector according to any one of claims 1 to 9, wherein the 3'UTR sequence of the PGRN includes the sequence of SEQ ID NO: 14, or a variant or homolog thereof having at least 90% sequence identity with SEQ ID NO: 14 .
  11. The AAV vector according to any one of claims 1 to 10, wherein the polynucleotide coding sequence includes a signal sequence derived from hGH.
  12. The AAV vector according to claim 11, wherein the hGH signal sequence includes the sequence of SEQ ID NO: 9 or a variant or homolog thereof having at least 90% sequence identity with SEQ ID NO: 9 .
  13. The AAV vector according to claim 4 or 5, wherein the codon-optimized sequence includes the sequence of SEQ ID NO : 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a variant or homolog thereof having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
  14. The AAV vector according to any one of claims 1 to 13, wherein the synapsin promoter comprises the sequence of SEQ ID NO: 15 or a variant or homolog thereof having at least 90% sequence identity with SEQ ID NO: 15 .
  15. The AAV vector according to any one of claims 1 to 14, wherein the AAV vector has the AAV9 serotype.
  16. The AAV vector according to any one of claims 1 to 15, wherein the nucleic acid sequence of the AAV vector has at least 90 % sequence identity with sequence number 17.
  17. A pharmaceutical composition comprising an AAV vector according to any one of claims 1 to 16, and one or more pharmaceutically or physiologically acceptable carriers, excipients, and/or diluents.
  18. A pharmaceutical composition according to claim 17, for use in the treatment of neurological disorders in subjects requiring such treatment.
  19. The pharmaceutical composition for use according to claim 18, wherein the neurological disorder is frontotemporal dementia (FTD), neuronal ceroid lipofuscinosis (NCL11), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, or Alzheimer's disease.
  20. The pharmaceutical composition for use according to claim 18 or 19, wherein the subject is (i) a heterozygous, homozygous, or compound heterozygous GRN mutation, (ii) suffering from a sporadic neurological disorder, and/or (iii) having a PGRN level lower than physiologically normal.

Description

(Field of Invention) The present invention relates to the field of genetically modified viral vectors. In particular, the present invention relates to genetically modified viral vectors suitable for in vivo delivery of therapeutic genes. (Background of the invention) Frontotemporal dementia (FTD) is the second most common form of dementia in patients under 65 years of age who exhibit symptoms. This dementia is characterized by changes in behavior, personality, and language, causing great distress to the patient, their family, and friends. Approximately 30% of FTD patients have a family history of dementia, and nearly 20% of these have loss-of-function (LoF) mutations in the GRN gene, which codes for the protein progranulin. Children with homozygous GRN mutations develop neuronal ceroid lipofuscinosis (NCL11), a lysosomal storage disorder characterized by neurodegeneration and blindness (JDRohrer et al., (2015b) Lancet Neurol. 14:253-262). Adults with heterozygous LOF mutations develop adult-onset FTD, which involves lysosomal storage lesions in cortical neurons of the frontal and temporal lobes, as well as TDP-43 inclusion body formation. MRI scans of GRN mutation carriers before the onset of the disease show structural differences in the temporal and parietal lobes approximately 15 years before the predicted clinical onset (Rohrer et al., cited above). Currently, there are no effective treatments that significantly alter the progression of any form of FTD, amyotrophic lateral sclerosis (ALS), or any form of neurodegenerative disease. The only medications prescribed for FTD are those to alleviate the effects of depression (antidepressants) or to reduce the effects of problematic behaviors (major tranquilizers), which often have serious and harmful short-term and long-term side effects. Progranulin (PGRN) is a secreted glycoprotein that has important nutrient effects on neurons, suppresses inflammation, and plays a crucial role in autophagy (B.P. Chitramuthu et al., (2017) Brain. 140(12):3081-3104). Progranulin is produced by microglia and neurons, but is taken up by all cell types. Upon binding to its receptor, sorbitol, on the cell membrane, progranulin is transported by endosomes to lysosomes, where it is broken down into multiple granulins (C.J. Holler et al., (2017) eNeuro. 18:4). One of these granulins acts as a chaperone for the protease cathepsin D, promoting lysosomal acidification and effective proteolysis. Lysosomal defects inhibit autophagy, leading to the accumulation of TDP-43 in the nucleus and cytoplasmic inclusions. GRN-/- mice exhibit subtle behavioral phenotypes (reduced social dominance) and do not show TDP-43 proteinopathy or neuronal loss, even when developing lipofuscinosis, reactive gliosis, and lysosomal abnormalities (A.W. Arrant et al., (2018) J. Neurosci. 38(9):2341-2358). Selective knockout of GRN from neurons in mice (T.L. Petkau et al., (2017) Neurobiol Dis. 106:14-22) or partial knockout from microglia (T.L. Petkau et al., (2017) J. Neuroinflammation. 14(1):225) reduced PGRN levels by approximately 50%, but did not result in any single phenotype or detectable disease state. This indicates that both cell types are capable of secreting sufficient progranulin to provide cross-collection throughout the tissue. Despite the lack of a suitable animal model, previous studies using viral vectors to deliver PGRN have shown promise. Studies showing that mouse GRN genes delivered via adeno-associated virus 1 (AAV1) and intraparenchymally injected into 12-month-old GRN-/- mice reduced lipofuscinosis and inflammation suggest that progranulin supplementation may reverse the disease's progression (A.E. Arrant et al., (2017) Brain. 140(5):1447-1465; A.E. Arrant et al., (2018) cited above). However, another study using human GRN delivered via AAV9 and AAV4 at approximately 33 times higher doses reported significant hippocampal degeneration with T cell infiltration in GRN-/- mice and non-transgenic mice after 3 months (D.A. Amado et al., doi: https://doi.org/10.1101/308692). Recent, remarkable successes in trials of AAV9-SMN in type I spinal muscular atrophy (SMA) have spurred the search for gene therapy for neurological disorders. SMA is caused by homozygous deletion of the survival motor neuron gene (SMN). These children are born with muscle weakness, and 95% require mechanical ventilation before the age of two, leading to progressive debilitation and the inability to sit, crawl, stand, or speak. Among 15 patients who received a single dose of AAV9-SMN between 1 and 7 months of age, none required mechanical ventilation, 14 of the 15 showed significant muscle strength, were able to stand or walk without assistance, and all were able to speak (J.R. Mendell et al., (2017) N. Engl. J. Med. 377(18):1713-1722). However, high doses of intravenous AAV9-SMN have been reported to be toxic in non-human primates and miniature pigs (C. Hinderer et al., (2018) Hum. Gene Ther. 29:285-298), and therefore care